Name: assessment of vascular regeneration in the cns using the mouse retina
Uploaded: 2014-06-23T18:45:00
Description: Watch this Scientific Journal Video about Assessment of Vascular Regeneration in the CNS Using the Mouse Retina at JoVE.com

PS는 망막 세포 생물학에있는 캐나다 연구 의자와 알콘 연구소의 새로운 탐정 상을 보유하고있다. 이 작품은 건강 연구의 캐나다 연구소 (221478)에서 교부금에 의해 지원되었다, 캐나다 당뇨병 협회 (OG​​ -3 - 11-3329 - PS), 캐나다 자연 과학 및 공학 연구위원회 (418637)와 재단 파이팅 실명 캐나다. 지원은 RESEAU 드 공들인 엉 상테 드 라 비전 뒤 퀘벡에 의해 제공되었다.

윤리 문 : 모든 동물 실험은 비전과 안과 안과에서 동물의 사용 및 비전 연구 및 동물 관리의 캐나다 협의회 (ARVO) 문에 연구를위한 협회에 의해 설립 된 동물 보호 지침을 준수합니다. 1. 산소 유도 망막 병증 (OIR) <ol><li> 기록 P0로 마우스 새끼의 생년월일. </li><li> 적절한 체중 범위를 보장하기 위해 O 2에 진입에 동물의 모든 무게를 기록한다. 참고 : P17의 ...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+selection+for+different+mtDNA+genotypes+in+heteroplasmic+mice.">Tissue-specific selection for different mtDNA genotypes in heteroplasmic mice.</a> Nat Genet. 16, 93-95 (1997).</li><li>Mattapallil, M. 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=The+Rd8+mutation+of+the+Crb1+gene+is+present+in+vendor+lines+of+C57BL/6N+mice+and+embryonic+stem+cells,+and+confounds+ocular+induced+mutant+phenotypes.">The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes.</a> Investigative ophthalmolog., &amp; visual science. 53, 2921-2927 (2012).</li></ol>

허혈성 신경 조직의 새로운 건강한 혈관의 성장을 촉진하는 가장 효과적인 방법은 무엇입니까? 그것은 방해하고 혈관 재성장을 자연적으로 발생하는 가속 치료 유효합니까? 허혈성 망막 병증이나 뇌졸중과 같은 신경 허혈성 병변에서 혈관 변성 감소 된 신경 세포의 기능을 35 ~ 38과 연결되어 있습니다. 따라서 질병의 초기 / 즉시 세그먼트 동안 지역의 미세 순환을 복직, 초기 부상에 대응...

CNS의 발달, 신경, 면역 세포 및 혈관에 걸쳐 적절한 조직 관류를 보장하고 감각 정보 1-5의 송신을 허용하도록 현저 결합 네트워크를 확립한다. 부족한 조직의 산소 및 손상 대사 공급하고있는 혈관 시스템의 결과의 분석은 점점 더 신경 퇴행성 질환 (6)의 발병 기전에 중요한 기여로 인식되고 있습니다. 혈관 강하 및 뇌 내의 신경 혈관 기기의 열화는, 예를 들어, 혈관성 치매, 뇌 7의 흰색 물질의 혈관 병변과 동맥 및 소형 선박 8 협착 알츠하이머 질환과 관련이 있습니다. 또, 손상된 혈관 장벽 기능은 다발성 경화증 (9) 및 근 위축성 측삭 경화증 (10)을 올릴 것으로 생각된다. 눈부신,이 프로토콜에 설명 된 망막 모델에 직접 관련이당뇨병 성 망막 병증 (11)과 미숙아 망막 병증 (12)과 같은 질병은 13 초 혈관 변성의 위상을 특징으로한다. 신경 혈관 망막에 계속되는 허혈성 스트레스 가능성이 다시 임명하다 산소와 에너지 공급 14 ~ 16에 대한 보상 반응으로 발생한 과도하고 병적 인 혈관 신생의 두 번째 단계를 트리거합니다. 질병의 진행에 중심 허혈성 스트레스를 극복하는 매력적인 전략은 특별히 신경 망막 (그림 2와 3)의 허혈 영역에서 기능 혈관 네트워크를 복원하는 것입니다. 제어 혈관 반응을 자극하는 직관적 안티 - VEGFs 같은 항 혈관 신생 치료가 적합한 치료로 간주되는 조건으로 건너 올 수 있습니다. 그러나,이 방법의 유효성에 대한 증거가 설치된다. 예를 들어, ischem에서 &quot;생리와 같은&quot;혈관 재성장을 강화IC의 망막 병증은 우아 내피 전구 세포 (17),식이 ω-3 고도 불포화 지방산을 증가 다른 혈관 신생 인자 (18), 골수 전구 세포 (19)의 주입, NADPH 산화 효소에 의한 세포 사멸 (20)의 억제의 뮐러 세포 발현 된 VEGF에 의한 하향 조절 억제의 도입을 통해 입증되었습니다 산 입구 (21), 트립토판의 tRNA 합성 효소 (22)의 카르 복실 말단 단편, 그리고 glial 세포 (23)의 보호를위한 VEGF 또는 FGF-2의 직접적인 관리와 치료. 또한, 우리는 허혈성 망막 병증 이러한 Semaphorins 또는 Netrins 같은 고전의 연결을 유도 신호를 변조하는 망막 내에서 건강한 혈관의 혈관 재생을 촉진하고 결과적으로 병적 인 혈관 신생 (24, 25)를 감소 시킨다는 것을 증명하고있다. 직접 임상 관련성의, 전술 한 동물 연구의 몇 가지 증거를 제공하는 혈관 재 추진망막증의 조기 허혈성 단계 동안 생성 가능성이 상당히 허혈성 부담의 감소를 통해, 시력을 위협 사전 망막 신생 혈관 (19), (23, 24), (26)을 감소시킬 수있다. 기능 혈관의 재생을 자극하는 치료 전략을 고안하는 것은 혈관 생물학을위한 중요한 과제로 남아. 여기에서 우리는 망막 내 혈​​관의 재성장을 조절하는 전략을 탐구하는 산소에 의한 망막 병증 (OIR)의 마우스 모델을 사용하는 실험 시스템을 설명합니다. 스미스 등에 의해 개발 1994. (27),이 모델은 인간의 증식 망막 병증에 대한 프록시 역할을하며 실내 순환 O 2 장력 (그림에 P12까지 75 %의 O 2 P7 마우스 새끼를 노출 이후에 새끼를 다시 도입 구성 1). 이 패러다임은 느슨하게 미숙아는 통풍이 시나리오를 모방O 2. 고농도 산소에 마우스 새끼의 노출은 망막 모세 혈관 및 미세 혈관의 변성을 유발하고, 최대 VO 영역이 후 48 시간 (P9)에 도달하지만, 일반적으로, P12에서 O 2 종료에 따라 평가 혈관 폐색의 재현 영역 (VO)를 산출 O 2 28에 노출. 마우스에서 무 혈관의 VO 영역은 자발적으로 실내 공기에 다시 소개 다음 주에 걸쳐 재생 결국 VO 지역은 완전히 (그림 2) 다시 혈관이 있습니다. OIR을 실시 생쥐의 실내 공기에 재 도입은 일반적으로 항 혈관 신생 치료 패러다임의 효능을 결정하기 위해 평가된다 사전 망막 혈관 신생 (NV) (P17에서 최대)을 불러 일으킨다. 그것의 순수한 형태, OIR 모델은 산소 - 유도 된 혈관 변성을 평가하고 파괴 사전 망막 신생 혈관 29-31의 범위를 결정하기 위해 높은 재현성 및 정량화 도구를 제공한다. <p claSS = &quot;jove_content는&quot;&gt; CNS의 혈관 재생을 조절하는 다양한 답사의 치료 패러다임이 약물 화합물, 유전자 치료, 유전자 삭제 등의 사용을 포함하여 OIR 모델을 사용하여 조사 할 수있다. 혈관 재성장에 영향을 미칠 수있는 특정 방법의 성향은 단계별 P12 (고농도 산소 종료 후 최대 VO)와 P17 (최대 NV) 사이의 창에서 평가된다. 병적 NV의 치료 결과의 평가는 신속하고 용이하게 병렬로 결정될 수있다 철저히 스탈 및 동료 30, 31에 의해 설명되었다. 여기에서 우리는 약물 화합물, 미래의 치료제, 바이러스 성 벡터에 의해 신경 망막 내에서 생리적 재관류 술의 변조를 조사하거나 유전자 변형 또는 녹아웃 마우스에서 후보 유전자의 영향을 연구하기 위해 간단한 단계별 절차를 제공합니다.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">C57Bl/6 mice ((Other strains may be used; angiogenic response varies from one strain to the other)</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:639px;">CD1 nursing mothers</td>
			<td style="width:181px;">Vendor of choice</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Operating Scissors straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">14192</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Dissecting Scissors straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">14393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Vannas Eye Scissors</td>
			<td style="width:181px;">Harvard Apparatus</td>
			<td style="width:175px;">72-8483</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Iris Forceps, curved, serrated</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">15915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Brushes 362R size 0</td>
			<td style="width:181px;">Dynasty</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:639px;">Dumont Forceps #3; straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">500338</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Surgical Blade, size 10</td>
			<td style="width:181px;">Bard-Parker</td>
			<td style="width:175px;">371110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Rhodamine Griffonia (Bandeiraea) Simplicifolia Lectin I</td>
			<td style="width:181px;">Vector Laboratories, Inc</td>
			<td style="width:175px;">RL-1102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Microscope slides</td>
			<td style="width:181px;">VWR</td>
			<td style="width:175px;">16004-368</td>
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			<td height="42" style="height:42px;width:639px;">Fluoromount G</td>
			<td style="width:181px;">Electron Microscopy Sciences</td>
			<td style="width:175px;">17984-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Zeiss Axio Observer Z1 Inverted Phase and Fluorescence Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:639px;">Leica MZ9.5 Stereomicroscope</td>
			<td rowspan="2" style="width:181px;">Leica</td>
			<td rowspan="2" style="width:175px;"></td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:639px;">Fluorescein isothicyanate-dextran, 70000</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:175px;">46945</td>
			<td></td>
		</tr>
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</table>

assessment of vascular regeneration in the cns using the mouse retina

설치류 망막 아마도 중추 신경계 (CNS) 신경 혈관 내 상호 작용을 조사 할 수있는 가장 접근 포유류 시스템이다. 그것은 점점 인식되고 그 알츠하이머, 다발성 경화증, 혈관 손상의 근 위축성 측삭 경화증 본 요소 등 여러 가지 신경 퇴행성 질환. 또한, 소아 및 가능 연령 인구 (각각 미숙아 및 당뇨 망막 병증,)에서 실명의 가장 눈에 띄는 원인은 생리적 혈관 재성장의 혈관 변성과 실패에 의해 특징입니다. 이 기술 문서의 목적은 망막에 CNS의 혈관 재생을 연구하기 위해 상세한 프로토콜을 제공하는 것입니다. 방법은 허혈 손상 후에 혈관 성장의 실패로 이어질 분자 메커니즘을 규명하기 위해 사용될 수있다. 또한, 잠재적 인 치료 양식 건강한 혈관 plexuses을 가속화하고 복원하는 탐험 할 수있다. 연구 결과는 obtaineD 설명한 방법을 사용하여 당뇨병이나 미숙아 것과 허혈성 망막 병증에 대한 치료 적 수단을 제공 할 가능성이 CNS의 다른 혈관 질환에 유익있다.

OIR 모델은 널리 망막에 산소에 의한 혈관의 변성과 허혈에 의한 병적 인 혈관 신생을 연구하는 데 사용되며, 안구 질환 27, 29, 30에 대한 현재 사용중인 항 혈관 신생 치료의 개발에 도움이되었습니다. 이 모델을 사용하여 얻은 연구 결과는 느슨하게 증식 성 당뇨 망막 병증 및 미숙아 (30)의 망막 병증 등의 허혈성 망막 병증으로 추정 할 수있다. 여기에 우리가 ...

Watch this Scientific Journal Video about Assessment of Vascular Regeneration in the CNS Using the Mouse Retina at JoVE.com

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

쥐의 망막은 오랫동안 뇌에 접근 창으로 인정을 받고있다. 이 기술 문서에서 우리는 허혈성 손상 후 중추 신경계 내에서 혈관 재생의 실패로 이어질 메커니즘을 연구하는 산소에 의한 망막 병증의 마우스 모델을 사용하는 프로토콜을 제공합니다. 기술 시스템은 망막과 중추 신경계 내에서 기능 혈관의 재성장을 촉진하는 전략을 탐구하는 무력화 할 수 있습니다.

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). ...

assessment-of-vascular-regeneration-in-the-cns-using-the-mouse-retina

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JoVE Journal

Neuroscience

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 ...

연구

신경과학

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

개발 마우스 망막의 생체내 Electroporation에

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

혈관을 조사하기 위해 신생아 마우스 망막의 전체 마운트 면역 형광 염색<em&gt; 생체 내</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

글루타민산 염과 GABA의 빠른 마이크로 이온 도입 : 시냅틱 통합을 조사하는 유용한 도구

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

각막 Micropocket 분석 : 마우스 눈에있는 혈관의 모델

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

이미징 CA<sup&gt; 2 +</sup마우스 망막의 광 수용체 콘 축삭 터미널에서&gt; 역학

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

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

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

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

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

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

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

성인 마우스 지 루트 신경 절의 Electroporation Vivo에서 조사 포유류 축 삭 재생:

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

설치류에서 시간적 처리의 평가 대 한 Interstimulus 간격의 힘

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

마우스 뇌의 수평 해마 조각

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...