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

נ.ב. מחזיק מחקר בקנדה קתדרת ביולוגיה של תא רשתית וניו פרס חוקר אלקון מכון המחקר. עבודה זו נתמכה על ידי מענקים מהמכון הקנדי לבריאות מחקר (221,478), איגוד הסוכרת הקנדי (OG-3-11-3,329-PS), מדעי הטבע והנדסת מועצת מחקר של קנדה (418,637) ועיוורון קרן הלחימה קנדה. תמיכה הייתה גם מסופקת על ידי Réseau דה משוכלל ונדיר en סנטה דה לה חזון du קוויבק.

שמירה על אתיקה: כל הניסויים בבעלי החיים שומרת הנחיות טיפול בבעלי החיים שהוקמו על ידי האגודה לחקר חזון העיניים הצהרה (ארוו) לשימוש בבעלי חיים ברפואת עיניים וחזון המחקר והמועצה הקנדית לטיפול בבעלי חיים. 1. רטינופתיה חמצן מו?...

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מהי הדרך היעילה ביותר להמרצת צמיחה של כלי חדשים ובריאים ברקמת העצבים איסכמי? האם זה טיפולי תקף להפריע ולהאיץ באופן טבעי לצמיחה מחודשת של כלי דם? בפתולוגיות נוירו איסכמי כגון Retinopathies או שבץ איסכמי, ניוון של כלי דם קשור לתפקוד עצבי מופחת 35-38. מכאן כדי להתמודד עם פג?...

לאורך פיתוח של מערכת העצבים המרכזית, עצבים, תאי מערכת החיסון וכלי דם להקים רשתות בשילוב להפליא כדי להבטיח זלוף רקמות נאות ולאפשר העברת מידע החושי 1-5. הפירוט של תוצאות מערכות כלי דם בחמצון מספיק רקמה ואספקה ​​מטבולים שנפרצו ומוכר יותר ויותר כתורם חשוב בפתוגנזה של מחלות ניווניות 6. נשירת כלי דם ואת ההידרדרות של יחידת העצבים וכלי הדם בתוך המוח, למשל, קשורה לדמנציה וסקולרית, נגעים של כלי דם של החומר הלבן של המוח 7 ומחלת אלצהיימר עם היצרות של כלי דם קטנים וarterioles 8. בנוסף, הוא חשב תפקוד מחסום כלי דם לקוי לתרום טרשת נפוצה 9 וטרשת לרוחב amyotrophic 10. של שייכות ישירה למודל הרשתית תואר בפרוטוקול זה, מסנוורמחלות כגון רטינופתיה סוכרתית 11 ורטינופתיה של פגות 12, 13 מתאפיינות בשלב של ניוון של כלי דם מוקדם. מתח איסכמי שהתפתח על רשתית העצבים וכלי הדם מפעיל שלב שני של neovascularization מוגזם ופתולוגי, כי סביר להניח שמקורו כתגובה מפצה לחמצן מחדש instate ואספקת אנרגיית 14-16. אסטרטגיה אטרקטיבית כדי להתגבר על מתח איסכמי שהוא מרכזי להתקדמות מחלה היא להחזיר את רשתות כלי דם פונקציונליות במיוחד באזורים איסכמי של נוירו הרשתית (איורים 2 ו -3). מעורר תגובת angiogenic מבוקר עלולה להתקל כמנוגד לאינטואיציה למצב בו טיפולים אנטי angiogenic כגון אנטי VEGFs נחשבים כטיפולים מותאמים. עם זאת, ראיות לתקפותה של גישה זו גוברת. לדוגמא, שיפור צמיחה מחודשת של כלי דם &quot;, כמו פיזיולוגית&quot; בischemRetinopathies ic הודגמה באלגנטיות באמצעות הקדמה של תאי האנדותל מבשר 17, עיכוב של downregulation המושרה VEGF-הביע תא Müller של גורמים האחרים angiogenic 18, הזרקה של אבות מיאלואידית 19, עיכוב של אפופטוזיס מושרה מונואמין NADPH 20, הגדלת שומן רב בלתי רווי בתזונה ω-3 צריכת חומצה 21, טיפול שבבר carboxyl-הטרמינל של synthetase tRNA טריפטופן 22, וניהול ישיר של VEGF או FGF-2 להגנה על תאי גליה 23. יתר על כן, אנו מראים כי ויסות אותות הנחיה עצביות קלאסיים כגון Semaphorins או Netrins בRetinopathies איסכמי מאיצה את התחדשות כלי דם של כלי בריאים בתוך הרשתית וכתוצאה מכך מפחיתה את היווצרות כלי דם פתולוגיים 24, 25. הרלוונטיות קלינית ישירה, כמה מהמחקרים בבעלי החיים האמורים לספק ראיות לכך שקידום מחדש של כלי דםדור בשלב המוקדם של איסכמי Retinopathies יכול להפחית באופן משמעותי neovascularization מסכנות ראייה מראש רשתית 19, 23, 24, 26, סביר להניח שבאמצעות הפחתת נטל איסכמי. גיבוש אסטרטגיות טיפוליות שלעורר התחדשות של כלי פונקציונליים עדיין מהוות אתגר משמעותי עבור ביולוגים של כלי דם. כאן אנו מתארים מערכת ניסיונית אשר מעסיקה את מודל העכבר של מחלת רשתית הנגרמת חמצן (OIR) לחקור אסטרטגיות כדי לווסת את צמיחה מחודשת של כלי דם בתוך הרשתית. שפותח על ידי סמית et al. ב -1994 27, מודל זה משמש כמדד לRetinopathies שגשוג אנושי וכולל חשיפה של גורי עכבר P7 75% O 2 עד P12 ולאחר מכן מחדש מציגים את הגורים לO חדר הסביבה 2-מתח (איור 1). פרדיגמה זו באופן רופף מחקה תרחיש שבו פג מאוורר<html> עם O 2. החשיפה של גורי עכבר לhyperoxia מעוררת התנוונות של נימים ונימי דם ברשתית, ותשואות אזור לשחזור של הרחבת כלי דם, הכחדה (VO) העריך בדרך כלל על יציאה מO 2 בP12, למרות שהוא הגיע לאזור VO המקסימאלי ב48 שעה (P9) לאחר חשיפה לO 2 28. בעכבר, אזורי VO avascular ספונטני להתחדש במהלך השבוע הבא מחדש מבוא לאוויר חדר וסופו של דבר אזורי VO הם לגמרי מחדש vascularized (איור 2). Re-מבוא לאוויר חדר של עכברים נתון OIR גם מעורר neovascularization מראש הרשתית (NV) (מקסימלי בP17) שהעריך בדרך כלל כדי לקבוע את היעילות של פרדיגמות טיפול אנטי angiogenic. בצורתו הטהורה ביותר, מודל OIR מספק כלי לשחזור וכמותיים מאוד להעריך ניוון של כלי דם נגרם על חמצן ולקבוע את מידת neovascularization מראש רשתית ההרסנית 29-31. ss = &quot;jove_content&quot;&gt; פרדיגמות טיפול explorative שונות לווסת התחדשות כלי דם במערכת העצבים המרכזית יכולה להיחקר באמצעות מודל OIR כולל שימוש בחומרים פרמקולוגיים, ריפוי גנטי, מחיקת גן ועוד. הנטייה של גישה שניתנה להשפיע לצמיחה מחודשת של כלי דם נבחנת צעד חכם בחלון בין P12 (VO המקסימאלי לאחר יציאה מhyperoxia) וP17 (NV המקסימאלי). הערכה של תוצאות טיפול בNV פתולוגיים ניתן לקבוע במהירות ובקלות במקביל ותוארה ביסודיות על ידי טל ועמיתיו 30, 31 ב. כאן אנו מספקים הליך של צעד אחר צעד פשוט כדי לחקור את האפנון של revascularization הפיזיולוגי בתוך הרשתית העצבית על ידי תרכובות תרופתית, תרופות פוטנציאליים, וקטורים ויראליים או ללמוד את ההשפעה של גנים מועמדים בעכברים מהונדסים או בנוקאאוט.

<table border="0" cellpadding="0" cellspacing="0" width="1224">
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		<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>
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			<td height="38" style="height:38px;width:639px;">CD1 nursing mothers</td>
			<td style="width:181px;">Vendor of choice</td>
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			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">14192</td>
			<td></td>
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		<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>
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			<td height="21" style="height:21px;width:639px;">Brushes 362R size 0</td>
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			<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>
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			<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>
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		<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>
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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>
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			<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>
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		<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>
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			<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>
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</table>

assessment of vascular regeneration in the cns using the mouse retina

רשתית המכרסמים היא אולי מערכת היונקים הנגיש ביותר בו כדי לחקור את יחסי גומלין עצבים וכלי דם במערכת העצבים המרכזית (CNS). זה יותר ויותר הכיר בכך שכמה מחלות ניווניות כגון אלצהיימר, טרשת נפוצה, ואלמנטי Amyotrophic Lateral Sclerosis הנוכחיים של פשרה כלי דם. בנוסף, הסיבות העיקריות לעיוורון באוכלוסיות גיל ילדים ועבודה (רטינופתיה של פגות ורטינופתיה סוכרתית, בהתאמה) מתאפיינות בניוון וכישלון כלי דם של צמיחה מחודשת של כלי דם פיזיולוגית. מטרת מאמר טכני זה היא לספק פרוטוקול מפורט ללמוד התחדשות כלי דם במערכת העצבים המרכזית ברשתית. השיטה יכולה להיות מועסק על מנת להבהיר את המנגנונים מולקולריים שיובילו לכישלון של צמיחת כלי דם לאחר פגיעת איסכמית. בנוסף, שיטות טיפוליות פוטנציאל להאיץ ולשחזר plexuses כלי דם בריא יכולות להיחקר. ממצאי obtaineד שימוש בגישה שתוארה עשויה לספק אפיקים טיפוליים לRetinopathies איסכמי כמו זו של סוכרת או פגות ואולי ירוויח מחלות כלי דם אחרות של מערכת העצבים המרכזית.

מודל OIR נעשה שימוש נרחב ללמוד ניוון של כלי דם נגרם על חמצן וneovascularization פתולוגיים נגרם איסכמיה ברשתית וכבר סייע בפיתוח טיפולים אנטי angiogenic מועסקים כיום למחלות עיניים 27, 29, 30. ממצאים שהתקבלו באמצעות מודל זה ניתן להסיק באופן רופף לRetinopathies איסכמי כגון רטינופתיה סוכרת?...

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

הרשתית המכרסם כבר זמן רב מוכרת כחלון נגיש למוח. במאמר זה טכני אנו מספקים פרוטוקול אשר מעסיק את מודל העכבר של מחלת רשתית הנגרמת חמצן כדי ללמוד את המנגנונים שיובילו לכישלון של התחדשות כלי דם במערכת העצבים המרכזית, לאחר פגיעה איסכמית. המערכת המתוארת גם ניתן לרתום לבחון אסטרטגיות לקידום צמיחה מחודשת של כלי דם פונקציונליים בתוך הרשתית ומערכת העצבים המרכזית.

הערכה של כלי דם התחדשות במערכת העצבים המרכזית באמצעות רשתית העכבר

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.

כימות של פעילות Cis-Regulatory אלמנטים ברשתית עכבר ידי electroporation Explant

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.

ב electroporation vivo של הרשתית עכבר פיתוח

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.

מכתים Immunofluorescent הר שלם של רשתית עכבר ילודים לחקר אנגיוגנזה<em&gt; בvivo</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.

מיקרו-Iontophoresis מהיר של גלוטמט וגאבא: כלי שימושי לחקירת האינטגרציה סינפטית

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.

קרנית Micropocket Assay: דגם של אנגיוגנזה בעין העכבר

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.

ההדמיה Ca<sup&gt; 2 +</sup&gt; Dynamics בסופי קון קולטי האור אקסון של רשתית העכבר

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.

שימוש בelectroretinogram כדי להעריך את התפקוד ברשתית מכרסמים וההשפעות המגנות של האיבר המרוחק איסכמי אכשור קד

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.

התחדשות האקסון בתרבית של החוקר: In Vivo אלקטרופורציה של העכבר למבוגרים העזוב שורש גנגליון

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.

הכוח של מרווח Interstimulus להערכה של זמני עיבוד בחולדות

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.

פרוסות היפוקמפוס אופקיות של מוח העכבר

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 ...