Biz video çekimi hazırlık düzenlemek için onu mükemmel teknik destek ve Mahesh Kumar Teli için Britta kaltwasser teşekkür etmek istiyorum.

1. Hayvanlar <ol><li> Deneyler laboratuvar hayvanlarının bakımı ve kullanımı için NIH kılavuzunda göre yürütülen ve yerel otoriteler tarafından onaylanmıştır. Tüm deneyler için, C57Bl6 / J yabani tip fareler, Apolipoprotein - / - (ApoE KO) ve (12-16 haftalık, 26-30 g vücut ağırlığı, deneysel grup başına 5-6 hayvan) SV129 fareler kullanılmıştır. </li></ol> 2. Renkli Latex ile Serebral Gemiler Boyanması <ol><li> EP bir tüp içinde bir 01:20 oranında lateks bileşiğin 0.5 ml (Pebeo, Fransa) ile 25 ul karbon siyah mürekkep (Herlitz, Almanya) oluşan bir karışım hazırlayın ve bir su banyosu içinde 37 ° C &#39;de karışım ısıtın. Hayvan bayıltmadan önce 28-30G bir iğne ile bir 2 ml şırınga içinde karışım toplayın. </li><li> Steril serum fizyolojik 1 ml içine papavarine hidroklorür toz 50 mg çözülür. Bir insülin şırınga çözümü toplayın. Başka steril insülin şırınga iğne kopartıp. Bir 20 ° C sonunda bu iğne yerleştirinm uzunluğunda PE10 tüp. Şimdi vazodilatasyon ve damarların uygun dolgu sağlamak için femoral ven yoluyla enjekte edilecek papavarine hidroklorür solüsyonu içeren insülin şırınga iğnesi bu PE10 tüp takın. </li><li> Başka bir iki insülin şırıngası tuzlu her birini içeren 1 ml hazırlayın. İnsülin iğneleri kesin bir yerde enjekte edilen sıvı düzgün teslimat sağlar. Ancak, lateks, alternatif hareketli daha geniş bir iğne ile 1 ml şırınga yüksek viskoziteye bağlı olarak kullanılmalıdır. </li><li> İşletme aşamasında yakın tüm şırınga ve steril kesme aletleri alın. Operasyon sahne etrafında bir cerrahi manifaturacı levha yayın. Işlem aşama yeterince ışıklı edilmesi ve gerekirse bir ameliyat mikroskobu kullanılabilir. </li><li> Şimdi bir C57Bl6 / J fare vücut ağırlığı ölçmek ve daha sonra% 5% 70 N 2 O ve% 30 O 2 izofluran buharlaşmış anestezi neden. Buharlaşmış izofluran% 1 ile anestezi devam edin. Uygun Positionin sonraekstremitelerin sırtını ve fiksasyon üzerinde fare g, anestezi derinliği değerlendirmek. Sonra sol femoral ven üzerinde cilt kesmek ve damar bulun. PE10 boru üzerine yerleştirilen iğne sivri ucu takın ve yavaş yavaş dakika başına 100 ul (50 mg / kg vücut ağırlığı) bir oranda papavarine hidroklorür çözelti enjekte edilir. </li><li> Enjeksiyonu takiben karın boşluğu ve delgi diyafram kesti. Bu zarar veya morarma olmadan kalp açığa kaburga kesin. Bir arter forsepsi ile inen torasik aorta klipsleyin. Venöz kan dışarı akmasını sağlamak için sağ atrium üzerinde keskin bir kesim olun. 18-20 sn üzerinde hafif basınç ile elle renkli lateks enjekte ardından, sol ventrikül içine serum fizyolojik 2 ml enjekte edilir. Önceden doldurulmuş şırıngalar birbiri ardına kullanın. Herhangi kabarcıklar enjekte kaçının. </li><li> Lateks enjeksiyonundan sonra 5 dakika için hayvan bırakın. Hayvan başını kesmek ve dikkatlice tüm beyin çıkarın. Gemi mimarisi ve corte kesmemeye iyi bakx kafatası kemiği ve meninks çıkarırken. Fotoğraf çekmek için% 4 PFA 6-8 ml&#39;lik 60 mm hücre kültür çanak beyin yerleştirin. Şeffaf çanak altında bir ölçüm cetvel yerleştirin. Operasyon mikroskobu bağlı bir kamera ile dorsal ve beynin ventral yüzeyi bir 25x büyütme fotoğraflar çekin. Resimler orijinal mesafeyi ölçmek amacıyla Petri kabı ve mikroskop objektif arasındaki 90 ° açıyla alınmalıdır. </li><li> Başka 4-5 hayvanlarda işlemi tekrarlayın. </li></ol> 3. Karbon Siyahı Mürekkepler karışımı ile Serebral Gemiler Boyanması <ol><li> Bizim protokol ile lateks bazlı serebral vasküler boyanma sonuçlarını karşılaştırmak için, bir 1.5 ml EP tüp Pelican Scribtol Schwarz mürekkep (CB2) 900 ul Herlitz Stempelfarbe mürekkep (CB1) 100 ul karışımı hazırlayın. Bir 01:09 oranında CB1 ve CB2 2 ml karışımının bir toplam hacim yapmak için iki EP tüp hazırlanır. Iki ayrı insülin şırıngası (1 içinde karışım toplayınml her). 37 ° C&#39;ye kadar ısınmasını şırınga kap ve su banyosu koyun Başka iki insülin şırıngaları salin 2 ml toplayın, ve de bir su banyosunda koyun. </li><li> Hayvan anestetize ve papavarine hidroklorür enjeksiyon için haricinde yukarıda açıklandığı gibi aynı prosedür tekrarlayın. Sol ventrikül içine 2 ml salaine enjeksiyonu takiben, karbon siyah mürekkep yerine renkli bir lateks 2 ml karışımı enjekte edilir. Ml başına 18-20 sn üzerinde hafif basınç ile elle enjeksiyonları gerçekleştirin. Enjeksiyonları öncesinde inen torasik aorta klipsleyin. </li><li> Hayvan Kurban, beyin çıkarın ve fotoğraf çekmek. </li><li> Yüzen beyin tamamen submerges kadar beynin uzun süreli korunması için, (% 5% 15 izledi ve ardından% 30) giderek artan konsantrasyon ile sukroz ile inkübasyon PFA gecede izledi% 4 beyin koymak. Renkli lateks ile perfüze hayvanların beyinlerinin de aynı şekilde muhafaza edilebilir. </li><li> GerçekleştirinBaşka 4-5 hayvanlarda prosedürü. Farklı genetik arka plan veya suşları nedeniyle serebral vasküler anatomi farkı karşılaştırmak için, sırasıyla, ApoE KO ve SV129 farelerin eşit sayıda protokol tekrarlayın. </li><li> Derin anestezi (cerrahi ayrıntılı bir açıklama bu makalenin kapsamı dışındadır altında MCA intraluminal tıkanıklığı standart bir protokole göre 45 dakika veya 90 dakika boyunca geçici fokal serebral iskemi neden, iskemik koşullarda vasküler anatominin incelenmesi; okuyucu Doeppner ve diğ., 2010) ifade edilir. Infarkt hacmi tanımlamak için reperfüzyon dönemi (1 gün veya 5 gün) planlanan sonunda, CB1 + CB2 mürekkepler yalnızca vasküler boyanma gerçekleştirmek ve 5-10 dakika süreyle 37 ° C&#39;de% 2 TTC çözeltisi ile tüm beyin leke . TTC mitokondrial enzimler (özellikle süksinat dehidrogenaz) tarafından kırmızı renkli formazan düşürülür. TTC boyama sonra, enfarkte doku unstai derin kırmızı metabolik olarak aktif doku lekeleri kalırkenned ve nedeniyle işlevsiz ve denatüre mitokondrial enzim beyaz görünür. Yukarıda tarif edildiği gibi aynı şekilde görüntü toplayın. </li></ol> 4. Serebral Vasküler Toprakları Çalışması <ol><li> Serebral damarlar, + CB2 mürekkepler ImageJ yazılım (görüntü işleme ve analiz için kullanılan bir Java tabanlı kamuya açık yazılım) ile analiz edilebilir renkli lateks veya CB1 ile beyin ya lekeli dorsal yüzeyinin görüntülerin gros anatomisinin analiz etmek. CB1 + CB2 perfüzyon ventral ve dorsal yüzeyleri hem tüm gemiler leke olurken renkli lateks ile perfüze edilen beyinler, dorsal yüzeyi üzerinde bulunan geminin boyanması değişik derecelerde gösterebilir. </li><li> ImageJ yazılım ile bir resim dosyası açın. ACA ve MCA arasındaki tüm anastomoz noktaları belirleyin. Anastomotik nokta damar çapı en dar ya da sırasıyla ACA en yakın dallanma noktaları ve MCA dalları arasındaki yarı mesafe, 2, yer olarak tanımlanır. <skadar&gt; segmente satırı aracı çizim yardım ve eklenti &quot;noktalı çizgi&quot; ile anastomoz noktaları işaretleyerek hayali bir çizgi (Anastomoz hattı) çizin. </li><li> Mm çaplı ayarlayın. Anastomoz hattı ve düz hat çizim aracı ve &quot;ölçü&quot; aracını kullanarak beynin frontal kutup kaudal 4 mm orta hat arasındaki mesafeyi ölçün. Kaudal frontal kutbundan 6 mm aynısını yapın. Hayvan başına 3-4 görüntüleri analiz eder ve ortalama değer hesaplayabilirsiniz. Serebrovasküler anatomi varyasyonu belirlemek için hayvanların farklı gruplar arasındaki değerleri karşılaştırın. C57Bl6 / J hayvanlarda, ACA ve MCA arasındaki Anastomoz hattı kaudal frontal kutbundan 6 mm den 4 mm daha yakın orta hat izlenebilmektedir. ApoE KO fareler bu açıdan anlamlı farklılık sunacak. Aksine, SV129 farelerde anastomoz hat ön kutup kaudale ileri ve 4 mm orta hattı hem de paralel ve 6 mm ortaya çıkacaktır. </li><li> Vasküler anatominin analiz etmek in iskemik durumlar, yukarıda belirtilen aynı hesaplamaları yapar. Sırasıyla, metabolik olarak aktif doku ve nekrotik unstained doku temsil kırmızı ve beyaz renkli doku marjı olarak infarktüsü sınır belirleyin. 4 mm ve beynin ön direğe yerden kaudal 6 mm orta hattan infarktüsü sınırın mesafeyi ölçün. Hayvan başına 3-4 görüntüleri ortalama değer toplayın. Farklı iskemi ve reperfüzyon dönemleri arasında sonuçları karşılaştırın. </li></ol>

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Çıkar çatışması ilan etti.

Bu belli bir basınç 2,3 ima için herhangi bir özel cihaz içermeyen manuel olarak enjeksiyon ile CB1 + CB2 perfüzyon yoğun bir eğitim olmadan ile başarılı bir şekilde gerçekleştirilebilir. Bizim protokol perfüzyon sonuçların heterojenliği azdır. 20 iskemik hayvanlar üzerinden sadece 1 16 iskemik olmayan hayvanların üzerinden hayvan ve 3 yetersiz perfüzyon göstermiştir. Bu durumlarda, damarların tıkanması neden tuzlu perfüzyon sırasında kabarcıklarının eklenmesi başarısız so...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Firma Adı </td><td> Katalog No </td></tr><tr><td> Scribtol Schwarz (CB2) </td><td> Pelican, Almanya </td><td> 221 135 </td></tr><tr><td> Stempelfarbe (CB1) </td><td> Herlitz PBS AG, Almanya </td><td> 10417202 </td></tr><tr><td> Gedeo Lateks </td><td> Pebeo, Fransa </td><td> 13042B </td></tr></tbody></table>

intravascular perfusion of carbon black ink allows reliable visualization of cerebral vessels

Beyin damarlarının anatomik yapısı beyin hemodinami için önemli bir belirleyici yanı sıra iskemik hakaret takip yaralanma şiddeti olduğunu. Beyin damarlarında dinamik çeşitli patofizyolojik devletler yanıt verir ve suşları arasındaki genetik manipülasyon koşullarında önemli farklılıklar sergilemektedir. Esasen, intrakraniyal damar boyama için güvenilir bir tekniktir iskemik inme patogenezinde çalışmak için esastır. Yakın zamana kadar, farklı teknikler bir dizi düşük viskoziteli reçine, araldit F enjeksiyonunu içeren beyin damarlarında, jelatin 2 veya siyah 3 karbon olmadan çeşitli boyalar 1 (yani karmin kırmızı, çini mürekkebi) veya lateks ile karıştırılır. Görselleştirmek için istihdam edilmiştir Asendan aorta yoluyla beyaz lateks bileşik Perfüzyon ilk Coyle ve Jokelainen 3 tarafından rapor edilmiştir. Maeda et al. 2 karbo ekleyerek değiştirilmiş olan protokolbeynin fizyolojik perfüzyon sonrası damarlarının gelişmiş kontrast görselleştirme için lateks bileşik n siyah mürekkep. Ancak, verimsiz perfüzyon ve gemilerin yetersiz dolum sık lateks bileşik 4 yüksek viskozitesi nedeniyle yaşanmaktadır. Bu nedenle, bir tekrarlanabilir bir şekilde 5 beyin damarlarında görselleştirmek için ticari olarak satılan iki karbon siyah mürekkep (CB1 ve CB2) oluşan bir karışım kullanılarak bir basit ve düşük maliyetli bir teknik tarif var. Biz lateks perfüzyon 5 ile karşılaştırıldığında daha yüksek bir yoğunlukta önemli ölçüde daha küçük beyin damarlarının boyama farelerde sonuç CB1 + CB2 ile bu perfüzyon göstermiştir. Burada, anterior (ACA) ve farklı genetik geçmişleri olan farelerde damar değişikliklerin incelenmesi orta serebral arter (MCA) arasındaki anastomoz noktaları belirlemek için protokol açıklar. Son olarak, CB1 birleştirerek farelerde bir geçici fokal serebral iskemi modelinde, bizim teknik olarak uygulanabileceğini göstermek +Iskemik yaralanma çeşitli derecelerde TTC boyama ile CB2-aracılı damar boyama.

Burada açıklanan protokol kemirgen beyin damarlarında geleneksel lateks bazlı görselleştirme teknik kısıtlamaların üstesinden gelmektedir. Şekil 1A renkli lateks bu aşağıdaki perfüzyon göstermektedir, ventral yüzeyi üzerinde sadece büyük damarlarda tüm dorsal yüzeyini renksiz bırakarak boyanmıştır. Sonuç da oldukça değişkendir. Beyin (veriler gösterilmemiştir) dorsal yüzeyinde görünür ACA ve MCA kısmi boyama altı gösterileri dışında sadece bir hayvan. Tersine, C...

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Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

Kemirgen serebrovasküler anatomi Analiz deneysel inme araştırmalarında önemli bir rol oynar. Bu çerçevede, renkli lateks ile intravasküler perfüzyon birkaç yıldır standart bir araç olarak kabul edilmiştir. Ancak, bu tekniğin tekrarlanabilirliği zayıflatmak farklı teknik sınırlamaları, ifade eder. Burada, tekrarlanabilir bir şekilde beyin damarlarının görselleştirmek için basit bir yöntem tarif. Yüksek kontrast görselleştirme ile beyin damarlarının yeterli dolgu sol ventrikül miyokard sonuçları ile iki ticari karbon siyah mürekkepler karışımı Enjeksiyon. Biz başarıyla farklı genetik geçmişleri olan farelerin serebral vasküler ülkelerle arasındaki anastomoz noktaları belirlemek için bu tekniği uyguladık. Farelerde infarkt hacimleri gözlemlemek ve analiz etmek için yaygın olarak kullanılan bir araçtır - Sonunda geminin boyanması için bu yeni ve basit bir yöntemdir Triphenyftetrazoliumchlorid (TTC) boyama ile kombine edilebilir olduğunu ifade vermek.

Karbon Siyah Mürekkep İntravasküler Perfüzyon Serebral Gemiler Güvenilir Görselleştirme verir

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The ...

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15.4K Görüntüleme.  University of Duisburg-Essen Medical School. Kemirgen serebrovasküler anatomi Analiz deneysel inme araştırmalarında önemli bir rol oynar. Bu çerçevede, renkli lateks ile intravasküler perfüzyon birkaç yıldır standart bir araç olarak kabul edilmiştir. Ancak, bu tekniğin tekrarlanabilirliği zayıflatmak farklı teknik sınırlamaları, ifade eder. Burada, tekrarlanabilir bir şekilde beyin damarlarının görselleştirmek için basit bir yöntem tarif. Yüksek kontrast görselleştirme ile beyin damarl...

Video: Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Endovasküler Dikiş Rat Orta Serebral Arter Tıkanıklığı Fokal Serebral İskemi Model

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Nörobilim

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

Hayvan İletişim Araştırmaları Otomatik Etkileşimli Video Oynatma

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

Whole Animal Perfusion Fixation for Rodents

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate 5,7. Often, changes in response to hypoxia begin before the tissue can be preserved 12. The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures 3. It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.

Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain.

Kemirgenler için Tüm Hayvan Perfüzyon Fiksasyon

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Kullanarak Fare Serebral Korteks ve Hippocampus Görselleştirme ve Dendritler ve Spine Genetik Manipülasyon<em&gt; Utero olarak</em&gt; Elektroporasyon

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Akut Hipokampal Dilimler içinde Synaptically-uyarılmış astroglial ve Nöronal Cevaplarının İkili Elektrofizyolojik Kayıtlar

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

Fare Beyin Gemiler saflaştırılması

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

İzolasyon ve Serebral Parankimal arteriollerin kanülasyonu

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

Yenidoğan Beyin içine Viral Parçacıklar ve Floresan mikro boncuklar intraserebroventriküler ve İntravasküler Enjeksiyon

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Microcoil stenoz ile hafif intrauterin hipoperfüzyon fare modeli

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Yalıtım taze insan beyin dokusundan beyin kapiller

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...