Bu çalışma kısmen NIH RO1-EY019951 (AAF), Körlük önlenmesi için Illinois Derneği (KİK, AAF), Körlük (RPB) Önleme Research Göz Hastalıkları Anabilim sınırsız para, NY ve RPB Tıp Öğrenci Bursu Grant tarafından desteklenmiştir (KİK).

1. Retina Hazırlık <ol><li> Fareyi göz tanıttılar. Tek elle, gözle görülebilecek şekilde göz kapakları açın. Diğer elinizle, kavisli bir forseps (kavisli yukarı bakacak şekilde) kullanarak, yörüngesinin üst ve alt yönleri üzerinde baskı uygulamak dünya çıkıntı kadar. Yavaşça gözün arka yönü de forseps kapatın ve göz tanıttılar sürekli bir hareket kaldırın. </li><li> Nötr% 10, en az 24 saat boyunca tamponlu formalin içine göz düzeltildi. </li><li> Küçük bir Petri kabındaki PBS çözüm yeri göz. </li><li> Büyük gözyaşları uyaran önlemek için özen, bir mikroskop altında dikkatle retina parçalara ayır. Diseksiyon makas ile kornea bir ilk kesim olun. Daha sonra, düz forseps ile kesim alanı tutarken, kornea çıkarmadan kornea ve sklera sınırı boyunca kesmek için makas kullanın. Dikkatle sklera ve uzak optik sinir doğru retina koroid kabuğu, ince kavisli ve düz forseps kullanma. Bu adım olarak sabır gerektirirhızlı bir şekilde serbest retina yırtıkları yol açabilir. Lens ve retina 16 emdiği iris ve pislikleri temizleyin. </li></ol> 2. Su yıkar <ol><li> 24 kuyulu bir yemek ve su (en az 45 mikron filtre ile süzüldü, yaklaşık 500 ul), ile kapakta yer retina. </li><li> , Her 30 dakika-1 saat en az 4-5 kez su değiştirin. </li><li> , Oda sıcaklığında nazik çalkalama ile su içinde bir gece bekletin. </li></ol> Not: kan damarlarından nöral retina katmanları ayrı yardımcı olarak yıkama adımlar çok önemlidir. 3. Tripsin Digest <ol><li> 37 0.1 M Tris tamponu (pH 7.8) içinde% 3 tripsin (Difco 1:250) solüsyonu ° C&#39;de herhangi bir çalkalama için yumuşak olan, su ve inkübe retina çıkarın. Doku parçalanması belirtileri (yaklaşık 1,5 saat) gösteren başladığında tripsin sindirmek tamamlanır. Not: hızlı sallayarak kaçınınBu damar zarar verebilir gibi. </li><li> Dikkatle, retina dokunmamak için bir mikroskop kullanarak iyi bir ayrı içine tripsin dışarı pipetle. Adım 4.3 mikroskop altında damarları, işlenirken Bu aşırı tripsin, daha sonra kullanılabilir. </li></ol> 4. Damarları ayırmak <ol><li> Retinaya filtrelenmiş su ekleyin. Daha sonra, gerekirse makas kullanarak, forseps ile iç sınırlayıcı membran kalkmasına çalışmayın. 5 dakika için hafif ajitasyon ile çalkalayın. Dikkatle, retina zarar vermemek için mikroskop altında su pipetle. </li><li> Yapışık retina dokusu, damar serbest ağ yardımcı olmak için su yıkama (her biri 5 dakika) için tekrarlayın. Yıkamadan sonra suda kalan hiçbir enkaz çok az olduğunda su yıkar tamamlanır. </li><li> Bir diseksiyon mikroskop altında dikkatli manipülasyon şimdi damarların ağ daha özgür ihtiyaç vardır. Aşağıda sırası veya ind kullanılabilecek birkaç yararlı teknikler vardırividually teknik tercihine göre istenen sonuca ulaşmak için: <ol class="lalpha"><li> 200 ul pipet kullanarak, dikkatli bir şekilde yavaşça sallanarak neden, gemi su üfleme, aşağı komşu gemilere su pipet ve. </li><li> 200 ul pipet kullanarak, pipet kat duvarları için aşağı tripsin yukarı pipet ve. Daha sonra, DİKKATLE olmayan vasküler doku yıkmak için bir kez tüm damar ağı ve aşağı yukarı pipetle. Not: yerine damar ağının kenarları daha optik sinir de pipet Merkezleme da ucu yapışmasını damar engelleyebilirsiniz. </li><li> Yavaşça olmayan vasküler bileşenleri çıkarmak için su dışında ince forseps ile damar kaldırın. </li><li> Yukarıdaki adımları işe yaramazsa, doku yıkmak yardımcı olur ve birkaç dakika ° C 37 inkübe retinaya tripsin ekleyin. Daha sonra, tripsin kaldırmak ve adımları 4.1-4.3 tekrarlayın. </li></ol></li></ol> &lt;strong&gt; Not: kat tripsin en gemiler (örneğin pipet, forseps) ile temas edecek tüm araçları emin olun. Bu ekipman damar yapışmasını önlemeye yardımcı olacaktır. 5. Damarları görselleştirme <ol><li> Temiz bir slayt üzerine bir damla su koyun. 200 ul pipet veya forseps kullanarak, suya sindirilir damarları aktarın. Emin olun vasküler düz montaj gelişeceğini edilir. Suyun yüzey gerilimi şimdi yarı saydam damar ağı açılmak için yardımcı olur. Doku açılmak değilse, ek su ekleyin ve hafifçe açığa çıkmasını kolaylaştırmak için slayt sallayın. </li><li> Bir kağıt havlu pipetleme veya kullanarak çok yavaş aşırı su çıkarmak. </li><li> Slaytlar tamamen kurumasını bekleyin. </li><li> PAS / hematoksilen veya H &amp; E boyama ile ya da uygun protokolü üzerinden slaytlar Leke. Not: PAS / hematoksilin damar duvarı ve hücre çekirdekleri tanımlamak için yararlıdır, H &amp; E, D için yararlıdırRBC emonstration de. </li><li> (Permount orta montaj) kurutmak ve monte edin. </li></ol>

<ol>
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Ophthalmol. 32, 487-498 (1949).</li><li>Podesta, F., 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=Bax+is+increased+in+the+retina+of+diabetic+subjects+and+is+associated+with+pericyte+apoptosis+in+vivo+and+in+vitro.">Bax is increased in the retina of diabetic subjects and is associated with pericyte apoptosis in vivo and in vitro.</a> Am. J. Pathol. 156 (10), 1025-1032 (2000).</li><li>Dagher, Z., 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=Studies+of+rat+and+human+retinas+predict+a+role+for+the+polyol+pathway+in+human+diabetic+retinopathy.">Studies of rat and human retinas predict a role for the polyol pathway in human diabetic retinopathy.</a> Diabetes. 53, 2404-2411 (2004).</li><li>Puro, D. 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Vis. 17, 3166-3174 (2011).</li><li>Li, Q., 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=Diabetic+eNOS-knockout+mice+develop+accelerated+retinopathy.">Diabetic eNOS-knockout mice develop accelerated retinopathy.</a> Invest. Ophthalmol. Vis. Sci. 51, 5240-5246 (2010).</li><li>Connor, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+oxygen-induced+retinopathy+in+the+mouse:+a+model+of+vessel+loss,+vessel+regrowth+and+pathological+angiogenesis.">Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis.</a> Nature Protocols. 4, 1565-1573 (2009).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Tripsin özet retinanın damar değerlendirmek için standart bir yöntemdir. Ne yazık ki, teknik olarak zor ve Doğru şekilde gerçekleştirilmemesi durumunda numune kaybı yüksek oranda neden olabilir. Ayrıca, işlem göz hastalıkları, yaygın olarak kullanılan bir genetik hayvan modellerinde bu tekniğin uygulanması sınırlandıran, farelerde özellikle zordur. Bu kağıt fare gözünde etkin ve sürekli yordamı gerçekleştirmek için nasıl rehberlik sağlar. Protokolde pek ç...

Retinanın damar görselleştirme, diyabetik retinopati gibi çeşitli göz hastalıklarının mekanizmaları incelemek için son derece önemli bir yaklaşımdır. Bu bir mikroanevrizmalar, kılcal dejenerasyon ve pericyte kaybı 8,9 dahil olmak üzere en erken vasküler anormallikler, değerlendirmenizi sağlar. Bugüne kadar, retina damar analiz etmek için geliştirilen çeşitli teknikler olmuştur. Çeşitli boyaların Perfüzyon damarları vurgulamak için kullanılmıştır, ama benzer kısıtlamaları paylaştı. Gemiler 1 kırılması ve zarar riskleri bir yüksek basınç, verilen sürece Enjeksiyon nadiren tüm retina damar vurgulamaktadır. Fluorofor etiketli G B4 isolectin (, I21413, Invitrogen, konjuge Alexa Fluor 594 1:100 seyreltme) ile vasküler endotel immün ve retina düz bağlar gemilerin genel mimarisi vurgulamak, ama kılcal damarların detaylı görüntülenmesi, bazal membran ve perisitler olmadan olabilir . Bu tarafından not edildiGemilerin periyodik asit-Schiff (PAS) veya hematoksilen ve eozin (H &amp; E) boyama 10 ile retinanın düz bağlar boyama vurgulanan olabilir Friedenwald. Ancak, boyama gemilere spesifik olmayan ve de olmayan vasküler doku vurgulanır, zor damarları ayırt etmek için yapmak. 1960&#39;larda, Cogan ve Kuwabara daha kolay retina 1 nonvasküler bileşenleri sindirerek retina damar görselleştirmek için yapılan tripsin sindirmek tekniği geliştirdi. O zamandan beri, tripsin sindirmek retinanın 2-5 damar analiz altın standart yöntem haline gelmiştir. Bununla birlikte, damar izole etmek için başka bir alternatif teknikler tarif edilmiştir dikkat etmek önemlidir. Ozmotik lizis kullanımı damar izole ve doku 11,12 biyokimyasal çalışmaları sağlamak için kullanılmıştır, ancak prosedür anatomik çalışma için birincil yöntem olarak kullanılmamıştır. Doku baskı yöntemi olmuşturmikrovasküler büyük bölümü izole etmek için kullanılan ve damar 13 elektrotonik mimarisi çalışma yeteneği sağlar. Teorik olarak, bu tekniği de gemilerin kalitesi yüksek olarak, anatomik değişiklikleri incelemek için kullanılabilir. Ancak, sadece damar ağı tüm bölümleri tespit edebilmektedir. Bu yöntemler tripsin sindirmek yerini alamaz rağmen, farklı avantajları ve eksiklikleri var ve bu konuda tamamlayıcı olduğunu not etmek önemlidir. Tripsin sindirmek yöntemi teknik olarak zor ve sürekli 6,7 gerçekleştirmek zordur. Ayrıca, bu istediği retina 6,7 genel vasküler mimarisi korumak için özellikle tripsin sindirmek, bir fare modeli özellikle zor olduğu görülmüştür. Sorunlar gerektiren, (1) yanı sıra, damar vasküler olmayan dokusu hem kaybına neden olan retinanın aşırı sindirilmesi, (2) altında yer sindirimen damar yatağının hasar, non-spesifik boyama yol açan damar gelen olmayan vasküler doku (3) kötü ayırma yol dönüşebilir geniş mekanik diseksiyonu. Çeşitli el yazmaları bu zorlukları ortaya koydu, ama hiçbiri onları 14,15 üstesinden gelmek için detaylı ve tutarlı bir protokol hazırladık. Bu yazının özellikle zor adımları kullanımı ile ilgili özel ipuçları ile fare ve sıçan retina üzerinde tripsin sindirmek gerçekleştirmek için teknik ayrıntılı bir adım adım yöntem tanıtacak. Şematik bir genel görünüşü Şekil 1&#39;de gösterilmiştir.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>10% neutral buffered formalin</td>
			<td>Fischer Scientific</td>
			<td>SF100-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin (1:250)</td>
			<td>Amresco</td>
			<td>0458-50G</td>
			<td>Make 3% trypsin in 0.1 M Tris</td>
		</tr>
		<tr>
			<td>TRIZMA base</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td>Create 0.1 M Tris Buffer (pH 7.8 @ 37 &deg;C)</td>
		</tr>
		<tr>
			<td>TRIZMA</td>
			<td>Sigma</td>
			<td>T3253-500G</td>
		</tr>
		<tr>
			<td>Steritop sterile vacuum bottle</td>
			<td>Millipore</td>
			<td>SCGPS05RE</td>
			<td>Create filtered water</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Any microscope that allows good visualization of the retina is adequate. (10x is sufficient with working distance of 24 mm)</td>
		</tr>
		<tr>
			<td>Straight scissors</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td>Cutting edge: 8 mm; Tip diameter: 0.2 mm; 10 cm</td>
		</tr>
		<tr>
			<td>Straight Forceps (Inox)</td>
			<td>Dumont #5</td>
			<td>11252-20</td>
			<td>Biologie tip; 0.05 x 0.02 mm; 11 cm</td>
		</tr>
		<tr>
			<td>Curved Forcepts (Dumostar)</td>
			<td>Dumont #7</td>
			<td>11297-10</td>
			<td>Biologie tip; 0.07 x 0.04 mm; 11.5 cm</td>
		</tr>
		<tr>
			<td>Dubnoff Metabolic Shaker Incubator</td>
			<td>Precision Scientific</td>
			<td>BDG59442</td>
			<td>Shaker optional</td>
		</tr>
		<tr>
			<td>24-well dish</td>
			<td>Falcon</td>
			<td>353047</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>VWR</td>
			<td>82027-132</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

trypsin digest protocol to analyze the retinal vasculature of a mouse model

Tripsin sindirmek retina damar 1-5 analiz etmek için altın standart bir yöntemdir. Bu retinanın vasküler olmayan bileşenlerin sindirim sonra birbirine vasküler kanalların bir iki boyutlu düz montaj oluşturarak karmaşık üç boyutlu retina kan damarları ve kılcal damarların tüm ağ görselleştirme sağlar. Bu böyle bir mikroanevrizmalar gibi çeşitli patolojik vasküler değişiklikler, kılcal dejenerasyon ve pericyte oranları anormal endotel çalışma sağlar. Ancak, yöntem hangi çünkü genetik manipülasyonlar 6,7 kolaylığı retina incelemek için en yaygın kullanılan hayvan modeli haline gelmiştir, özellikle farelerde, teknik olarak zordur. Fare gözünde, bu retinal kan damarlarının genel yapısını muhafaza ederken tamamen vasküler olmayan bileşenleri ortadan kaldırmak için özellikle zordur. Bugüne kadar, ayrıntılı olarak tripsin sindirmek teknik anlatılmaktadır edebiyat bir eksiklik olduğunu iFare n. Ayrıca zor adımlar sorun giderme ipuçları sağlarken Bu makale, fare retinada tripsin özet ayrıntılı bir adım adım yöntem sağlar.

Başarılı bir prosedür nihai ürün Şekil 2-4 de gösterildiği gibi mimari, PAS / hematoksilin ve H &amp; E ile boyandı ya da, muhafaza ile birlikte, fare retina damar sistemine ait bütün ağ düz bir montaj olup. Şekil 3&#39;te gösterildiği gibi, endotelyal hücreler ve perisitler açık bir farklılaşma görülebilir. Retina endotelyal hücrelerinin çekirdeklerindeki oval veya uzun ve damar duvarı tamamen içinde yer alır. Pericyte çekirdekleri küçük, küresel, yoğ...

Watch this Scientific Journal Video about Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model at JoVE.com

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Tripsin sindirmek retina damar analiz etmek için en sık kullanılan yöntemlerden biridir. Bu yazının gemilerin genel mimarisi korurken teknik optimize etmek ve olmayan vasküler doku kaldırmak için önemli değişiklikler de dahil olmak üzere, ayrıntılı olarak yöntem açıklanır.

Tripsin Digest Protokolü Fare Modeli Retina damarları Analiz

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

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Neuroscience

18.3K Görüntüleme.  Northwestern University Feinberg School of Medicine. Tripsin sindirmek retina damar analiz etmek için en sık kullanılan yöntemlerden biridir. Bu yazının gemilerin genel mimarisi korurken teknik optimize etmek ve olmayan vasküler doku kaldırmak için önemli değişiklikler de dahil olmak üzere, ayrıntılı olarak yöntem açıklanır.

Video: Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Retina Bölüm morfometrik Analizleri

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Nörobilim

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

Rat Beyin gelen Serebral Yüzey damarları ve İlişkili Beyin zarları ve Diseksiyon ve Koroid Pleksus bir Görsel Açıklama

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Fare Retina Ganglion Hücreleri Synaptic ve Yapay iletkenlikleri Dinamik Kelepçe Uygulama

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

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.

Kornea mikrocep Testi: Fare Eye Angiogenezis Bir Model

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

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

Fare Beyin Çeşitli Bölgelerinde Telomeraz Aktivite: Radyoaktif olmayan Telomeraz Amplifikasyon Protokolü tekrarlayın (TRAP) Deneyi

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

Video Görüntüleme ve Spatiotemporal Haritalar Fare Gastrointestinal Motilite Analiz

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

Protocol for Isolating the Mouse Circle of Willis

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and hypothalamus that supplies blood to the brain and surrounding structures. It has been implicated in several cerebrovascular disorders, including cerebral amyloid angiopathy (CAA)-associated vasculopathies, intracranial atherosclerosis and intracranial aneurysms. Studies of the molecular mechanisms underlying these diseases for the identification of novel drug targets for their prevention require animal models. Some of these models may be transgenic, whereas others will involve isolation of the cerebro-vasculature, including the CoW.The method described here is suitable for CoW isolation in any mouse lineage and has considerable potential for screening (expression of genes, protein production, posttranslational protein modifications, secretome analysis, etc.) studies on the large vessels of the mouse cerebro-vasculature. It can also be used for ex vivo studies, by adapting the organ bath system developed for isolated mouse olfactory arteries.

We describe here a reproducible protocol for isolating the mouse circle of Willis.

Willis Fare Çemberi Ayırma Protokolü

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Immünhistokimya için fare retinal Cryo-bölümler hazırlanması

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Değişkenliği Sınırlamak için Retinal Ven Tıkanıklığı Fare Modelinin Optimizasyonu

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...

Using a Bipolar Electrode to Create a Temporal Lobe Epilepsy Mouse Model by Electrical Kindling of the Amygdala

The amygdala is one of the most common origins of seizures, and the amygdala mouse model is essential for the illustration of epilepsy. However, few studies have described the experimental protocol in detail. This paper illustrates the whole process of amygdala electrical kindling epilepsy model making, with the introduction of a method of bipolar electrode fabrication. This electrode can both stimulate and record, reducing brain injury caused by implanting separate electrodes for stimulation and recording. For long-term electroencephalogram (EEG) recording purposes, slip rings were used to eliminate the record interruption caused by cable tangles and falling off.
After periodic stimulation (60 Hz, 1 s every 15 min) of the basolateral amygdala (AP: 1.67 mm, L: 2.7 mm, V: 4.9 mm) for 19.83 &plusmn; 5.742 times, full kindling was observed in six mice (defined as induction of three continuous grade V episodes classified by Racine's scale). An intracranial EEG was recorded throughout the entire kindling process, and an epileptic discharge in the amygdala lasting 20-70 s was observed after kindling. Therefore, this is a robust protocol for modeling epilepsy originating from the amygdala, and the method is suitable for revealing the role of the amygdala in temporal lobe epilepsy. This research contributes to future studies on the mechanisms of mesial temporal lobe epilepsy and novel antiepileptogenic drugs.

The amygdala plays a key role in temporal lobe epilepsy, which originates in and propagates from this structure. This article provides a detailed description of the fabrication of deep brain electrodes with both recording and stimulating functions. It introduces a model of medial temporal lobe epilepsy originating from the amygdala.

Amigdala'nın Elektriksel Çırası ile Temporal Lob Epilepsi Fare Modeli Oluşturmak için Bipolar Elektrot Kullanma

The amygdala is one of the most common origins of seizures, and the amygdala mouse model is essential for the illustration of epilepsy. However, few ...