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

PS heeft een Canada Research Chair in retinale Celbiologie en de Alcon Research Institute New Investigator Award. Dit werk werd ondersteund door subsidies van de Canadese Institutes of Health Research (221.478), de Canadese Diabetes Association (OG-3-11-3329-PS), de Natural Sciences and Engineering Research Council of Canada (418.637) en de Stichting Bestrijding Blindheid Canada. Ondersteuning werd ook door de Reseau de Recherche en Sante de la Vision du Quebec.

Ethiek verklaring: Alle dierproeven houdt zich het dier zorg richtlijnen die door de Vereniging voor Onderzoek naar Visie en Oogheelkunde (ARVO) verklaring voor het gebruik van dieren in Oogheelkundige en Vision Research en de Canadese Raad van Animal Care. 1. Zuurstof Induced retinopathie (OIR) <ol><li> Geboortedatum van muisjongen als P0 Record. </li><li> Noteer alle gewichten van de dieren bij binnenkomst in O 2 om een voldoende gewicht bereik te garanderen. ...

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L., Joly, S., Hardy, P., Chemtob, S., Lachapelle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+oxygen+and+light+on+the+structure+and+function+of+the+neonatal+rat+retina.">The effect of oxygen and light on the structure and function of the neonatal rat retina.</a> Doc Ophthalmol. 118, 37-54 (2009).</li><li>Chopp, M., Zhang, Z. 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Wat is de meest effectieve manier om de groei van nieuwe gezonde schepen in ischemisch zenuwweefsel stimuleren? Is het therapeutisch geldig te beïnvloeden en versnellen van nature voorkomende vasculaire hergroei? In de neuro-ischemische aandoeningen zoals ischemische retinopathie of een beroerte, wordt vasculaire degeneratie geassocieerd met een verminderde neuronale functie 35-38. Vandaar vroeg letsel tegen te gaan, het herstel van regionale micro-circulatie tijdens de onmiddellijke / vroege segment van de ...

Gedurende CNS ontwikkeling, zenuwen, immuuncellen en bloedvaten vast opmerkelijk gekoppeld netwerken om adequate weefsel perfusie zorgen en laat de transmissie van sensorische informatie 1-5. De verdeling van vasculaire systemen resulteert in onvoldoende zuurstofvoorziening van de weefsels en gecompromitteerde metabole vraag en wordt steeds meer erkend als een belangrijke bijdrage aan de pathogenese van neurodegeneratieve ziekten 6. Vasculaire uitval en de verslechtering van de neurovasculaire eenheid binnen de hersenen, bijvoorbeeld, is geassocieerd met vasculaire dementie, vasculaire laesies van de witte stof van de hersenen 7 en Alzheimer met stenose van arteriolen en kleine vaten 8. Bovendien wordt verslechterde vasculaire barrièrefunctie gedacht multiple sclerose 9 en amyotrofe laterale sclerose 10 dragen. Van direct belang zijn voor de retinale model beschreven in dit protocol, verblindendeziekten zoals diabetische retinopathie en 11 retinopathie van prematuriteit 12, 13 gekenmerkt door een fase van vroege vasculaire degeneratie. De daaropvolgende ischemische belasting van de neurovasculaire netvlies veroorzaakt een tweede fase van overmatig en pathologische neovascularisatie die waarschijnlijk afkomstig als compenserende reactie opnieuw instate zuurstof en energievoorziening 14-16. Een aantrekkelijke strategie om de ischemische stress die centraal ziekteprogressie overwinnen is om functionele vasculaire netwerken herstellen specifiek in de ischemische gebieden van de neuro-retina (figuren 2 en 3). Veroorzakend een gecontroleerde angiogene respons kan overkomen contra-intuïtief een aandoening waarbij anti-angiogene behandelingen zoals anti-VEGF&#39;s worden beschouwd als aangepast behandelingen. Toch bewijs voor de geldigheid van deze aanpak is montage. Bijvoorbeeld, het verbeteren van &quot;fysiologische-achtige&quot; vasculaire hergroei in ischemic retinopathiën is elegant aangetoond door middel van introductie van endotheliale voorlopercellen 17, remming van Müller-cel tot expressie VEGF geïnduceerde downregulatie van andere angiogene factoren 18, injectie van myeloïde voorlopercellen 19, remming van NADPH oxidase geïnduceerde apoptose 20, het verhogen van de voeding ω-3 meervoudig onverzadigde vetzuren zuur inname 21 behandeling met een carboxyl-terminaal fragment van tryptofaan tRNA synthetase 22 en directe toediening van VEGF of FGF-2 voor de bescherming van gliacellen 23. Bovendien hebben we aangetoond dat het moduleren van neuronale klassieke Praktische signalen zoals Semaforines of netrins in ischemische retinopathie versnelt vasculaire regeneratie van gezonde vaten in het netvlies en daardoor vermindert pathologische angiogenèse 24, 25. Van directe klinische relevantie, een aantal van de hiervoor genoemde dierlijke studies tonen aan dat het bevorderen van vasculaire regeneratie tijdens de vroege fase van ischemische retinopathie kan een aanzienlijke besparing-zicht bedreigen pre-retinale neovascularisatie 19, 23, 24, 26, waarschijnlijk door de vermindering van ischemische belasting. Het bedenken van therapeutische strategieën die regeneratie van functionele schepen te stimuleren blijft een belangrijke uitdaging voor vasculaire biologen. Hier beschrijven we een experimenteel systeem dat de muis-model van zuurstof-geïnduceerde retinopathie (OIR) om strategieën te verkennen om vasculaire hergroei te moduleren in het netvlies in dienst heeft. Ontwikkeld door Smith et al.. In 1994 27, dit model fungeert als een proxy voor de menselijke proliferatieve retinopathieën en bestaat uit het blootstellen van P7 muis pups tot 75% O 2 tot P12 en vervolgens opnieuw introduceren van de pups tot omgevingstemperatuur O 2-spanning (Figuur 1). Dit paradigma losjes bootst een scenario waarbij een te vroeg geboren baby wordt geventileerdmet O 2. De blootstelling van de muis pups te hyperoxie veroorzaakt degeneratie van het netvlies haarvaten en microvasculature, en levert een reproduceerbare gebied van vaso-uitwissen (VO) doorgaans aan bij het ​​verlaten van O 2 op P12, hoewel maximale VO gebied na wordt bereikt bij 48 uur (P9) blootstelling aan O2 28. In de muis, de avasculaire VO zones spontaan te regenereren in de loop van de week na herintroductie om ruimte lucht en uiteindelijk VO zones zijn volledig opnieuw gevasculariseerd (figuur 2). Herintroductie kamerlucht van muizen blootgesteld aan OIR lokt pre-retinale neovascularisatie (NV) (maximaal bij P17) die meestal wordt beoordeeld om de effectiviteit van anti-angiogene behandeling paradigma bepalen. In zijn zuiverste vorm, de OIR model geeft een zeer reproduceerbare en kwantificeerbare instrument om zuurstof-geïnduceerde vasculaire degeneratie beoordelen en bepalen de omvang van de destructieve pre-retinale neovascularisatie 29-31. <p class = &quot;jove_content&quot;&gt; Diverse exploratieve behandeling paradigma dat CNS vasculaire regeneratie moduleren kan worden onderzocht met behulp van de OIR model inclusief het gebruik van farmacologische stoffen, gentherapie, gendeletie en meer. De neiging van een bepaalde aanpak van vasculaire hergroei te beïnvloeden wordt beoordeeld stapsgewijs in het venster tussen P12 (maximale VO na verlaten hyperoxia) en P17 (maximale NV). Evaluatie van de resultaten van de behandeling op pathologische NV kan snel en eenvoudig worden bepaald parallel en is grondig beschreven door Stahl en collega&#39;s 30, 31. Hier geven we een eenvoudige stap-voor-stap procedure om de modulatie van fysiologische revascularisatie onderzoeken in de neurale retina van farmacologische verbindingen potentiële therapeutica, virale vectoren of de invloed van kandidaat genen in transgene of knockout-muizen te bestuderen.

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			<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>
			<td></td>
		</tr>
		<tr height="42">
			<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>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
		</tr>
		<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>
	</tbody>
</table>

assessment of vascular regeneration in the cns using the mouse retina

Het knaagdier netvlies is misschien wel de meest toegankelijke van zoogdieren systeem waarin om neurovasculaire samenspel binnen het centrale zenuwstelsel (CNS) te onderzoeken. Het wordt steeds meer erkend dat verscheidene neurodegeneratieve ziekten zoals de ziekte van Alzheimer, multiple sclerose en amyotrofe laterale sclerose aanwezige elementen van vasculaire compromis. Bovendien zijn de belangrijkste oorzaken van blindheid bij kinderen en werkende leeftijd populaties (prematuren retinopathie en diabetische retinopathie, respectievelijk) gekenmerkt door vasculaire degeneratie en falen van fysiologische vasculaire hergroei. Het doel van dit technische document is een gedetailleerd protocol bieden aan CNS vasculaire regeneratie te bestuderen in het netvlies. De werkwijze kan worden toegepast om de moleculaire mechanismen die leiden tot falen van vasculaire groei na ischemisch letsel te helderen. Daarnaast kunnen potentiële therapeutische modaliteiten te versnellen en het herstel van gezonde vasculaire plexus worden onderzocht. Bevindingen obtaineD De beschreven benadering kan therapeutische middelen voor ischemische retinopathie zoals diabetes of prematuriteit en eventueel gebruikmaken andere vasculaire aandoeningen van het CZS.

De OIR model wordt veel gebruikt om zuurstof geïnduceerde vasculaire degeneratie en ischemie geïnduceerde pathologische neovascularisatie studeren in de retina en is bij de ontwikkeling van de momenteel toegepaste anti-angiogene behandelingen voor oogziekten 27, 29, 30 zijn. Bevindingen verkregen met behulp van dit model kan los worden geëxtrapoleerd naar ischemische retinopathie zoals proliferatieve diabetische retinopathie en retinopathie van prematuriteit 30. Hier...

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

Het knaagdier netvlies is lang erkend als een toegankelijke venster naar de hersenen. In dit technisch document bieden we een protocol dat het muismodel van zuurstof geïnduceerde retinopathie van de mechanismen die leiden tot het falen van vasculaire regeneratie binnen het centrale zenuwstelsel na ischemische schade te bestuderen in dienst heeft. Het beschreven systeem kan ook worden ingezet om strategieën te ontwikkelen om hergroei van functionele bloedvaten te bevorderen binnen het netvlies en CNS.

Beoordeling van de Vasculaire Regeneratie in het CZS met de muis Retina

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

Research

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.

Kwantificeren van de activiteit van Cis-Regulatorische elementen in de muis Retina door Explantatie Elektroporatie

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

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.

In vivo Elektroporatie van de ontwikkelingslanden Mouse Retina

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.

Hele Mount immunofluorescentiekleuring van de Neonatale Mouse Retina naar Angiogenese Onderzoek<em&gt; In vivo</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.

Snel Micro-iontoforese van glutamaat en GABA: een nuttig instrument om Synaptic Integratie Onderzoek

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.

De Cornea micropocket Analyse: Een model van angiogenese in de muis Eye

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.

Imaging Ca<sup&gt; 2+</sup&gt; Dynamiek in Cone fotoreceptorcellen Axon Terminals van de Mouse Retina

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.

Met behulp van de electroretinogram om Function beoordelen in het knaagdier Retina en de beschermende effecten van Remote Limb Ischemische preconditionering

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.

Behandelende zoogdieren Axon regeneratie: In Vivo Electroporation van volwassen muis achterwortelganglia 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 ...

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.

De kracht van Interstimulus Interval voor de beoordeling van Temporal Processing in knaagdieren

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.

Horizontale Hippocampal Plakjes van de Hersenen van de Muis

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 ...