Bartly J. Mondino MD, 줄스 스타 인 안구 연구소, UCLA의 이사;, 그리고 라미 네즈 - 솔리스, 재키 선배 화이트와 Sanger 연구소, 웰컴 트러스트 게놈 캠퍼스에서 쟌 Estabel 실명 방지를위한 연구. 이 연구는 안과 및 Visual 연구에서 동물의 사용에 대한 ARVO 보호 정책을 준수합니다.

1. 무딘 절개 : 고정되지 않은 표본에서 마우스 눈의 핵의 제거 <ol><li> 노출과 뒤쪽 세계 (안구) 표면에 대한 액세스를 개선하기 위해 눈꺼풀를 분열. </li><li> 궤도의 (이하) 세계 (눈 소켓) 뒤에 곡선 드레스 forcep를 놓습니다. Mahajan Sharptip 드레싱 forcep은 (재료 및 시약 표 참조)이 단계를 쉽게하기 위해 뾰족한 팁을 사용자 정의 악기입니다. </li><li> forcep을 닫고 세계를 쥐어 피하려면 조심하면서 전 세계 뒤에 궤도 결합 조직과 시신경을 파악. </li><li> 부드럽게 위쪽으로 forcep을 뽑아 궤도에서 눈알을 뽑아 내다. 흰색 스레드와 같은 조직은 시신경입니다. </li><li> PBS의 눈을 놓으십시오. 30 게이지 바늘을 사용하여, 하나의 구멍이 안구에 바늘 1~2밀리미터를 삽입하여 limbus에 바로 뒤쪽 상처를합니다. 이 안구 조직을 침투하기가 달리없는 등 글 루타 알데히드 등의 fixatives는,,, 실내 직접 수행 할 수 있습니다적이있는 눈. 이 정착액 등 paraformaldehyde 또는 알코올 등, 관심 안구 조직에 diffusing 할 경우이 찔린 상처를 만들 필요하지 않을 수 있습니다. </li><li> 즉시 침지 고정 용 정착액으로 전체 눈을 놓습니다. </li></ol> 2. 샤프 해부 : 마우스 눈 다음 재관류 고정의 핵의 제거 <ol><li> 거리에 세계에서 눈꺼풀을 보유하는 곡선 colibri forcep을 사용합니다. </li><li> 전 세계에 평행 동양 곡선 Westcott의 분해 가위, 궤도의 뒷면을 향해 목표로하는 동안. </li><li> 하부, 중간, 우수한, 그리고 측면 측면에서 세계를 둘러싸고있는 고정 결합 조직을 잘라. </li><li> 세계 뒤에 곡선 forcep을 놓고 세계에 눌러없이 결합 조직을 파악하고, 눈을 명백히하다 앞으로 당긴다. 궤도 결합 조직은 관류 고정에서 치열한이기 때문에, 궤도 조직의 추가 절단 완전히 글로브를 공개 할 필요가있을 수 있습니다5. </li></ol> 3. 고정 및 패키징 고정 조직의 배송은 적절한 라벨 및 포장을 포함하여 기관 및 우편 서비스 요구 사항을 준수해야합니다. 다음 프로토콜은 주위 온도에서 비 전염성 생물 샘플을 출하의 예입니다. 이 3 포장 층 있지만 클래스 A / B 생물학적 물질 또는 액체 질소 라벨이 필요합니다. <ol><li> 고정 짧은 경우 최소 3-ML의 정착액, 포스트 정착액 버퍼로 5 ML 유리 섬광 유리 병에 눈을 넣습니다. 유리 병의 뚜껑에있는 추적 번호를 작성하고 Parafilm로 밀봉. </li><li> 생물학적 샘플로 장소 섬광 튜브는 배송 컨테이너를 승인했습니다. 샘플이 배치되는 밀폐 용기, 흡수 중간 계층 및 보호 외부 층 : 우리 연구소에서 예를 들어, 승인 컨테이너는 3 층이 필요합니다. </li><li> 버블 랩 패키지로 한 다음 응용 프로그램에 컨테이너를 놓고적절한 서류와 함께 roved 실험실 배송 컨테이너. </li></ol> 4. 대표 결과 눈의 조직 학적 시험은 Hematoxylin과 Eosin의 착색, 효소 표현 감지, 전송 전자 현미경, 그리고 immunohistochemistry 등의 다양한 방법으로 수행 할 수 있습니다. 4 % paraformaldehyde에 침적 고정 후, 조직은 파라핀과 마이크로톰에 sectioned에 포함되었다. 높은 처리량 phenotyping, 우리는 학생 - 시신경 부분을 분석이 눈에 샘플 모든 조직 유형 (그림 1). 조직의 제는 lacZ 표현 (그림 2), 세포 세포 소기관의 전송 전자 현미경 (그림 3) 및 항체 (그림 4)와 특정 분자의 표현을 검토했다. 우리의 표현형 검사 방법에서는 (latera 코에 대해 (중간 안각)과 측두엽과 눈의 방향을 유지하기 위해 중요하지리터 안각)면. 눈의 방향이 필요한 경우 최소한 두 가지 옵션이 있습니다. 핵의 제거 후, ​​우리는 휴대용 소작 장치와 세시 위치에 시간적 각막에 빛 소작을 적용했습니다. 병변은 조직 학적 소견에 분명하고 조직 학적 잉크와 같은 처리하는 동안 손실되지 않습니다. 단점은 그 소작 손해 시험을위한 중요한 조직이 될 수있는 각막이다. 다른 옵션은 extraocular 근육 삽입을 식별 할 수있는 숙련 된 해부학자가 필요합니다. stereomicroscope에서 하부 우수한 경사 근육 삽입의 식별이 세계의 하부 및 뛰어난 및 측면을 표시합니다. 3 중 오리엔테이션 방법으로는 오른쪽 또는 왼쪽 눈으로 표본을 추적하는 것이 중요합니다. 함께이 sectioning하기 전에 정확한 전 방향을 할 수 있습니다. <img src="/files/ftp_upload/3184/3184fig1.jpg" alt="그림 1" /> 1 그림. 마우스 눈 조직 학적 이미지는 afte<html> R 원격 획득. 마우스 눈은 4 % paraformaldehyde로 관류 고정 후 날카로운 절개로 enucleated했다. Hematoxylin과 Eosin 물들 A.의 학생 - 시신경이 섹션은 모든 조직 substructures의 보존을 보여줍니다. 시신경이 없으면, ON. , IPL, 내부 plexiform 레이어, INL, 내부 핵 계층, OPL, 외부 plexiform 레이어, ONL, 외부 RGC, 망막 신경절 세포 : 일반 망막의 (녹색 규모의 바 = 500 마이크론) B. 높은 배율의 전망은 판상 구조를 보여줍니다 핵 레이어 (photoreceptor 세포) (화살표), OS, photoreceptor 외부 세그먼트; RPE, 망막 안료 상피, C, 맥락막, photoreceptor 내부 세그먼트 (화살표 머리)입니다. (녹색 규모 바 = 50 마이크론) C.는 정상적인 망막에 비해,이 표본은 photoreceptor 변성에 얇은 때문입니다. ONL는 있으며, OS는 (화살표) 완전히 결석입니다. (녹색 규모 줄이 = 50 마이크론) &quot;ALT =&quot;그림 2 &quot;/&gt; 유전자 변형 쥐 그림 2. lacZ의 표현. 눈 (5 MM K3Fe (CN) 6, 5 MM K4Fe (CN) 6, 2 MM MgCl2, 0.02 % NP40 무딘 절개로 enucleated 1 MG / ML X-여자 솔루션에 몰두했다 37의 밤 및 0.1 % 나트륨 deoxycholate) ° C. 눈은 30 분 마이크로톰에 sectioned 2 % formaldehyde/0.2 %의 글 루타 알데히드에서 수정되었습니다. X-여자 제품 (파란색) 레이블 : A. Ciliary 상피, B. 각막 상피와 C. 망막의 망막 신경절 세포 (RGC) 계층. INL, 내부 핵 계층, ONL, 바깥 쪽 핵 층. <img src="/files/ftp_upload/3184/3184fig3.jpg" alt="그림 3" /> 그림 3. 망막 색소 상피 (RPE)의 전송 전자 현미경. 찔린 상처의 무딘 절개과 창조 후, 눈을 집중했다는 0.1M 나트륨 인산염 버퍼에 2.5 %의 Paraformaldehyde / 2.5 %의 글 루타 알데히드에 고정. 눈이 secon했다darily, 1 % 오스뮴의 textroxide에서 수정 서서히 건조하고, Spurr의 수지에 포함. 조직은 90 나노 미터에서 sectioned이고 Formvar로 덮여 구리 슬롯 격자에 배치하고 전송 전자 현미경을 사용하여 이미징. 현미경은 photoreceptor 외부 neurosensory 망막의 세그먼트, 망막 안료 상피 내 melanosomes (M), 그리고 Bruch의 멤브레인을 보여줍니다. <img src="/files/ftp_upload/3184/3184fig4.jpg" alt="그림 4" /> 4 그림. 마우스 유리체에서 초과 산화물 dismutase-3 Immunohistochemical 라벨 (녹색). 눈이 무딘 절개로 enucleated와 4 % paraformaldehyde에 침적 고정을 받았습니다되었습니다. 그들은 마이크로톰을 사용하여 파라핀과 sectioned에 포함되었습니다. 1시 50분을 희석 조직 섹션은 토끼 polyclonal SOD3 항혈청과 incubated되었습니다. SOD3 표현은 염소 안티 - 토끼 IgG (H + L) - 알렉사 형석 488 복합 차 항체를 사용하여 감지되었습니다. SOD3의 라벨은 GRE에 표시됩니다ko 페이지를 참조하십시오.

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</ol>

관심 없음 충돌이 선언 없습니다.

대부분의 유전자 변형 마우스는 눈을 검사하지 않는 실험실에 존재합니다. 우리 비디오 기술은 눈 작은 경험이있는 실험실에서 조직 획득을 최적화 할 수 원격 수술 기술 이전을위한 간단한 표준화 된 방법을 보여줍니다. 이 비디오 기술은 의미있는 비교 phenotypic와 분자 연구를 방지 비 표준화 조직 수집 및 고정 방법에 의한 전문 사이트 제한된 수의를 사용하는 것입니다 높은 처리량 phenotyping에 주요 함정을 극복하는 데 도움이됩니다. 새로운 원격 실험실과 기술자와 품질 관리를 설정하고 유지하기 위해, 그것은 시작과 정기적으로 연구를 통해 야생 형 눈을 포함하는 것이 중요합니다. 우리는 또한 여러 genotypes에서 여러 눈 등 각각의 눈에 고유 한 식별 번호를 할당 할 수있는 웹 기반 시스템으로, 전송 때 추적 시스템이 중요하다는 것을 발견했다. 높은 수준의 고정 및 후속 조직 섹션을 얻을 수 있지만재관류 - 고정 동물, 우리는 고정되지 않은 동물로부터 enucleated 표본은 대부분의 형태학 및 분자 연구를위한 충분한 것을 찾을 수 있습니다. 또한, 고정되지 않은 동물의 눈의 취득은 다른 소스에서 높은 처리량 연구에서 처리 할 수​​ 상당히 쉽게 할 수 있습니다. 유전자 변형 마우스는 지역 phenotyping이 귀중한 자원의 대부분을 만드는에 대한 인간의 안과 질환, 10, 11 및 원격 조직 획득을 조사하기위한 중요한 악기입니다. 조직 절개 및 공유를위한 비슷한 전략의 응용 프로그램이 아닌 안과 조직의 높은 처리량 phenotyping에 중요한 영향을 미칠 수 있습니다.

<table border="1"><tbody><tr><th> 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 코멘트 </th></tr><tr><td> 구부러진 드레싱 Forcep </td><td> Storz Ophthalmics </td><td> E1408 </td><td></td></tr><tr><td> Mahajan Sharptip 드레싱 forcep </td><td> Storz Ophthalmics </td><td> E1406 (REF SP7-64520) </td><td></td></tr><tr><td> 곡선 Westcott의 가위 </td><td> Storz Ophthalmics </td><td> E3321 WH </td><td></td></tr><tr><td> 15 ° BD 비버 Microsurgical 블레이드 </td><td> Becton - 디킨슨 </td><td> 374881 </td><td></td></tr><tr><td> 0.22 미세 Castroviejo Suturing의 포셉 </td><td> Storz Ophthalmics </td><td> E1805 </td><td></td></tr><tr><td> 0.12 Colibri 포셉 </td><td> Storz Ophthalmics </td><td> 132분의 2 </td><td></td></tr><tr><td> 30 게이지 바늘 </td><td> Becton - 디킨슨 </td><td> 305128 </td><td></td></tr><tr><td> 바이오 해저드 메일 서버 </td><td> 어부 </td><td> 03-523-4 </td><td></td></tr><tr><td> Parafilm </td><td> 어부 </td><td> 13-374-10 </td><td></td></tr><tr><td> 유리 섬광 튜브 </td><td> 휘튼 </td><td> 4500413033 </td><td></td></tr><tr><td> PBS, pH를 7.4 </td><td> Invitrogen </td><td> 70011-044 </td><td></td></tr><tr><td> 16 % Paraformaldehyde </td><td> 전자 현미경 과학 </td><td> 15,700 </td><td></td></tr><tr><td> 0.1M 나트륨 인산염 버퍼에 2.5 %의 Paraformaldehyde / 2.5 %의 글 루타 알데히드 </td><td> 전자 현미경 과학 </td><td> 15700 및 16300 </td><td> 실험실에서 혼합 </td></tr><tr><td> 50 % 글 루타 알데히드 </td><td> 전자 현미경 과학 </td><td> 16,300 </td><td></td></tr><tr><td> 0.25 % Formvar </td><td> 전자 현미경 과학 </td><td> 15,810 </td><td></td></tr><tr><td> 순경슬롯 그리드 당 </td><td> 전자 현미경 과학 </td><td> M2010-CR </td><td></td></tr><tr><td> 4 % 오스뮴의 Tetroxide </td><td> 전자 현미경 과학 </td><td> 19,140 </td><td></td></tr><tr><td> 안티 SOD3 항체 </td><td> Abcam </td><td> Ab21974 </td><td></td></tr><tr><td> 염소 안티 - 토끼 알렉사 형석 488 </td><td> Invitrogen </td><td> A11070 </td><td></td></tr><tr><td> Spurr의 삽입 수지 </td><td> 전자 현미경 과학 </td><td> 14,300 </td><td></td></tr></tbody></table>

mouse eye enucleation for remote high-throughput phenotyping

마우스 눈은 인간의 안과 질환의 병진 연구의 중요한 유전 모델입니다. 이러한 황반 변성, photoreceptor 변성, 백내장, 녹내장, retinoblastoma, 그리고 당뇨병 성 망막증과 같은 인간에 실명을 야기하는 안구 질환을 치료는 1-5 대부분의 유전자 변형과 녹아웃 마우스가 아닌 안과 질환을 연구하기 위해 실험실에 의해 생성되었습니다. 유전자 변형 마우스에 recapitulated하지만, 한 장기 시스템 사이의 유전 절약은 동일한 유전자의 많은도 안구 개발과 질병에 역할을 할 수 있습니다 제안합니다. 따라서,이 마우스는 눈에 새로운 유전자형 - 표현형의 상관 관계를 발견하기위한 중요한 자원을 나타냅니다. 이 마우스는 전 세계에 흩어져 있기 때문에, 취득, 유지 관리 및 효율적 비용 효율적인 방법으로 표현형을하기가 어렵습니다. 따라서, 대부분의 높은 처리량 안과 phenotyping 화면은 라이브 쥐에 눈을 검토하기 위해 안과 전문 현장에서 필요로 몇 가지 위치로 제한됩니다. 6-9는 우리 연구실에서 개발 한 대체 접근 방식은 유전자 변형 마우스 눈 크고 작은 규모의 설문 조사에서 사용할 수있는 원격 조직 - 취득하는 방법입니다. 비디오 기반의 수술 기술 이전, 조직 고정 및 배송을위한 표준화 된 절차는 실험실 돌연변이 동물로부터 전체 눈을 수집하고 분자 및 형태학 phenotyping에 보내 할 수 있습니다. 이 비디오 문서에서는, 우리는 원격 phenotyping 분석을 위해 떼어 낸 및 재관류 모두 고정 마우스 눈을 명백히하다하고 전송하는 기술을 제시한다.

Watch this Scientific Journal Video about Mouse Eye Enucleation for Remote High-throughput Phenotyping at JoVE.com

Mouse Eye Enucleation for Remote High-throughput Phenotyping

분해 기술은 높은 처리량 화면에서 phenotyping을 수행 할 조직 고정에 대한 마우스 눈의 핵의 제거를 보여줍니다.

원격 높은 처리량 Phenotyping에 마우스 눈 핵의 제거

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

mouse-eye-enucleation-for-remote-high-throughput-phenotyping

Nanyang Technological University

Research

JoVE Journal

Medicine

20.3K 조회수.  University of Iowa. 분해 기술은 높은 처리량 화면에서 phenotyping을 수행 할 조직 고정에 대한 마우스 눈의 핵의 제거를 보여줍니다.

비디오: Mouse Eye Enucleation for Remote High-throughput Phenotyping

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

멀티 컬러 형광 기자 마우스를 사용하여 각막 세포의 리니지 추적하는 방법

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

연구

발생학

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

의 활동을 Quantifying CIS</emExplant Electroporation하여 마우스 망막에서> - 규제 요소

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

신경과학

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

연령 관련 황반변성의 마우스 모델에서 망막 색소 상피 병리를 평가하고 정량화하기 위한 프로토콜

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal ...

유전학

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

차세대 염기서열분석을 위한 뮤린 망막 내피 세포의 분리

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

광학 일관성 단층 촬영: 마우스 망막 신경 절 세포에서 Vivo에서 화상 진 찰

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

마우스 각막과 결막의 라이브 이미징을 위한 맞춤형 다광현미경 플랫폼

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

생체공학

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

초고 해상도 마우스 광학 일관성 단층 촬영 망막 유전자 치료 연구에서 안 구내 주사를 돕기 위해

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

의학

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 ...

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

Enucleation or the surgical removal of an eye can generally be considered as a model for nerve deafferentation. It provides a valuable tool to study the different aspects of visual, cross-modal and developmental plasticity along the mammalian visual system1-4.
Here, we demonstrate an elegant and straightforward technique for the removal of one or both eyes in the mouse, which is validated in mice of 20 days old up to adults. Briefly, a disinfected curved forceps is used to clamp the optic nerve behind the eye. Subsequently, circular movements are performed to constrict the optic nerve and remove the eyeball. The advantages of this technique are high reproducibility, minimal to no bleeding, rapid post-operative recovery and a very low learning threshold for the experimenter. Hence, a large amount of animals can be manipulated and processed with minimal amount of effort. The nature of the technique may induce slight damage to the retina during the procedure. This side effect makes this method less suitable as compared to Mahajan et al. (2011)5 if the goal is to collect and analyze retinal tissue. Also, our method is limited to post-eye opening ages (mouse: P10 - 13 onwards) since the eyeball needs to be displaced from the socket without removing the eyelids. The in vivo enucleation technique described in this manuscript has recently been successfully applied with minor modifications in rats and appears useful to study the afferent visual pathway of rodents in general.

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

The removal of eyes, also called enucleation, provides a useful strategy to study aspects of visual, cross-modal, and developmental plasticity along the mammalian visual system since it induces irreversible partial (monocular) or complete (binocular) vision loss. Here we describe a highly reproducible and straightforward approach to perform in vivo enucleation.

높은 재현성과 단순 명료 한 방법이 수행하는<em&gt; 생체</em눈 개봉 후 마우스에서&gt; 안구 탈핵

Enucleation or the surgical removal of an eye can generally be considered as a model for nerve deafferentation. It provides a valuable tool to study the ...

생물학

Microdissection of the Rodent Eye

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, to obtain the anterior and posterior eyecups. With this technique, the sub-parts of the eye, including the corneal tissue, neural tissue, retinal pigment epithelial (RPE) tissue, and lens, can be obtained for wholemounts, cryo-sectioning, and/or single-cell suspensions of a specific ocular tissue. The presence of the third eyelid presents unique and significant advantages, as it benefits the maintenance of the orientation of the eye, which is important for understanding eye physiology following any localized intervention or in studies involving ocular analysis relating to the eye&#39;s spatial topography.
In this method, we enucleated the eyeball at the socket along with the third eyelid by carefully and slowly cutting through the extraocular muscles and severing the optic nerve. The eyeball was pierced through the corneal limbus using a microblade.&#160;The incision was used as the point of entry, allowing for cutting along the corneal-scleral junction by inserting micro-scissors through the incision point. Small and continuous cuts along the circumference were made until the cups separated. These could be further dissected by gently peeling the translucent layer of the neural retina using Colibri suturing forceps to obtain the neural retina and RPE layers. Further, three/four equidistant cuts were made from the periphery perpendicularly to the optic center until the optic nerve was reached. This opened the hemispherical cups into a floret shape so that they fell flat and could be easily mounted. This technique has been used in our lab for corneal wholemounts and retinal sections. The presence of the third eyelid delineates the nasal-temporal orientation, which allows for the study of various cell therapy interventions post-transplantation and, thus, the targeted physiological validation vital for visualization and accurate representation in such studies.

This paper presents a protocol for ocular micro-dissection in rodents. The process involves the enucleation of the eyeball along with the nictitating membrane (i.e., the third eyelid). This is then followed by the separation of the posterior and anterior eye cups.

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, ...