Bu çalışma kısmen JH Zhang NIH hibe RO1NS053407 tarafından desteklenmiştir. Biz onun değerli katkıları için Sayın Damon Klebe teşekkür etmek istiyorum.

Tüm prosedürler Laboratuvar Hayvanlarının Bakımı ve Kullanımı için NIH Kılavuz uygun olarak yürütülmesi ve Loma Linda Üniversitesi Hayvan Bakım ve Kullanım Kurulu tarafından onaylanmıştır. 1. Cerrahi öncesi hazırlıklar Aseptik teknikler tüm cerrahi işlemler için tavsiye edilir. Stereotaktik cihazları dezenfekte ve cerrahi öncesinde steril cerrahi araçları hazırlamak. Tüm hayvan taşıma sırasında kişisel koruyucu ekipman (KKE) giyin. Hayvanın fizyolojik vücut ısısını korumak için ameliyat sırasında bir ısıtma yastığı kullanın. <ol><li> Bir üçlü ışın hayvan ölçeği kullanılarak 8-12 hafta eski fare tartılır. </li><li> Co-enjekte ketamin (100 mg / kg) ve ksilazin (10 mg / kg) intraperitoneal sonra anestezi için 7-10 dk (yeterli bir sedasyon için monitör) etkili olması için izin. </li><li> Bir termal battaniye üzerine fare yerleştirin ve kafa derisi traş. </li><li> Her iki göze oftalmik merhem uygulayın. </li><li> Güvenlihavayolu, hafifçe yanal dil hareket ve dikkatle stereotaktik cihaz üzerine fare kafası sabitleyin. Not: baş stereotaksik çerçeve temelinde yatay olarak korunmalıdır. </li><li> Betadine ile cerrahi alanında dezenfekte ve% 70 etanol ile yıkayın. Üç kez toplam Betadin ve% 70 etanol ile ve uygulamaları alternatif tekrarlayın. Pamuk uçlu aplikatör bu amaç için kullanılabilir. </li></ol> 2. Kan Enjeksiyon Modeli <ol><li> Bir # 10 Bistüri ile kafa derisi bir 1 cm uzunluğunda orta hat kesi olun. </li><li> Koronal ve sagittal sütür (bregma) ve dik kesişme noktasında ortaya koymak için, kafatası kapsayan yumuşak doku kaldırmak için pamuk uçlu aplikatör kullanın. </li><li> Enjeksiyon pompası üzerine Hamilton şırınga (250 ul) monte edin ve steriotaksik bregma üzerinde iğne (26 Gauge) yönlendirin. </li><li> Daha sonra, iğne 0,2 mm konumlandırmak için stereotaktik manipülatör kollar ayarlamak ön ve 2yanal sağa mm. At bu koordinatları bir 1 mm matkap ucu ile bir değişken devirli kullanarak küçük bir kafatası burr hole yapmak. </li><li> Hayvanın kuyruk askıya alınması ve% 70 etanol ile alt yüzeyi dezenfekte. </li><li> Delinme merkezi kuyruk steril bir iğne (örneğin 26 Gauge) ile arter ve heparinize olmayan kapiler tüp içine kan toplamak. </li><li> Hamilton şırıngası cam varil içine kılcal tüp hızla kan aktarın, sonra dalgıç yerleştirin. </li><li> Enjeksiyon pompası üzerine Hamilton şırınga içeren arteriyel kan artık 30 ul veya daha fazla tekrar takın ve konik artık görünür sadece kadar burr deliğinden iğne (sutur bakan eğimli kenar ile) sokun. </li><li> Bu noktadan itibaren önceden iğne 3 mm ve ventral 2 ul / dak &#39;lık bir hızla otolog kan 5 ul enjekte edilir. </li><li> Ilk enjeksiyon önceden derinlik de iğne 0.7 mm tamamlanmasından sonra. </li><li> Beklemek5 dk sonra sağ striatum içine kan 25 ul enjekte. </li><li> İkinci enjeksiyon tamamlanmasından sonra, 1 mm / dak &#39;lık bir hızla o çekilmeden önce, ilave 10 dakika boyunca bir konumda iğne bırakın. </li><li> Kemik balmumu ile burr hole Seal ve cilt sütüre. </li><li> Postoperatif analjezi için önceden ısıtılmış sıvılar (normal salin) subkütan olarak buprenorfin 0.05 mg / kg enjekte. </li></ol> 3. Kollajenaz Enjeksiyon Modeli <ol><li> Kan enjeksiyonu modeli için açıklandığı gibi cerrahi öncesi hazırlıklar tamamlandıktan sonra, tekrar 1-4 adımları. </li><li> 0.5 ul tuzlu su içinde çözülmüş bakteri (clostridial) kolajenaz VII-S 0.075U ile Hamilton şırınga (10 ul) doldurun. Hava kabarcıklarının oluşumunu önleyiniz. </li><li> Enjeksiyon pompası üzerine Hamilton şırınga takın ve konik artık görünür sadece kadar burr deliğinden, iğne (26 Gauge) takın. </li><li> Ventral iğne 3,7 mm ilerletin ve kollajenaz 0.075U enjekte i2 ul / dak &#39;lık bir hızla nto doğru striatuma. </li><li> Enjeksiyon tamamlandığında, 1 mm / dak &#39;lık bir hızla o çekilmeden önce, ilave 10 dakika boyunca bir konumda iğne bırakın. </li><li> Kemik balmumu ile burr hole Seal ve cilt sütüre. </li><li> Önceden ısıtılmış post-operatif sıvılar içinde subkutan olarak buprenorfinin 0.05 mg / kg enjekte edilir. </li></ol> 4. Sham Operasyonu <ol><li> Kan enjeksiyonu modeli için açıklandığı gibi cerrahi öncesi hazırlıklar tamamlandıktan sonra, tekrar 1-4 adımları. </li><li> Burr deliğinden ventral iğne (26 Gauge) 3.7 mm takın. İğne 1 mm / dak &#39;lık bir hızla içe dönük edilmeden önce 10 dakika boyunca bir konumda kalması gerekmektedir. </li><li> Kemik balmumu ile burr hole Seal ve cilt sütüre. </li><li> Önceden ısıtılmış post-operatif sıvılar içinde subkutan olarak buprenorfinin 0.05 mg / kg enjekte edilir. </li></ol> 5. Temsilcisi Sonuçlar Deneysel intrastriatal kanama gibi morfolojik çağrıştırıyorkemirgenlerde sıra davranış değişiklikleri. Bu değişiklikler prosedür yeterli bir getirilmesini sağlamak için, ya da potansiyel tedavilerinin etkilerinin araştırılması için değerlendirilebilir. Hedeflenen bir beyin bölgesi (örneğin bazal gangliyon) içinde kanama oluşturma tekrarlanabilir bir yaklaşım için en önemli ve brüt veya histolojik olarak boyanan beyin dokularının (Şekil 1-2) üzerinde doğrulanabilir. Çeşitli davranışsal değerlendirmeler yoluyla ölçülebilir sensorimotor defisitler içinde bazal ganglion sonuçlar yaralanma. Köşe dönüş testi sonuçları deney sağ taraflı İSH&#39;lı, fareler (Şekil 3 A) sham cerrahi sonrası 24 ve 72 saatte hayvanlar ameliyat daha, engelli kontralateral (sol) yan Tortikollis ve uzak anlamlı olarak daha sık döndü, gösterdi. Ayrıca, yeterince vibrissaenin uyarılmasını takiben, bir yüzey üzerine engelli (sol) ön ayakları yere yeteneği, ön ayakları yerleştirme testi ile değerlendirildi. 24 ve 72 saat ameliyat sonrası, fareler altSağ taraflı ICH için jected sham operasyonu hayvanlara oranla daha az yerleşim gösterdi. Beyin ödemi ölçümü sıklıkla deneysel İSH&#39;lı beyin hasarı ölçüde ölçmek için kullanılır. Otolog kan (30 ul) veya bakteriyel kollajenaz intraserebral enjeksiyonu (0.075 U) önemli bir 24. ipsilateral korteks ve bazal ganglionlar beyin su içeriği artışı (Şekil 4 A) ve ameliyat sonrası 72 saat (Şekil 4 B) (yol açtı ) sham karşılaştırıldığında. Davranış testleri (Şekil 3) ve beyin ödemi (Şekil 4) kapsamının sonucu verilen hacimlerde kan ve kollajenaz enjeksiyon modelleri arasında fark göstermedi. <img alt="Şekil 1" src="/files/ftp_upload/4289/4289fig1.jpg" /> Şekil 1. Farelerde Modelleme ICH. (A) bregma anterior koronal beyin bölümü 0,2 mm basitleştirilmiş şematik pr göstermektedirotolog kan veya kollajenaz enjeksiyon yeri oposed. Lateral ventrikül LV işaretlenir. CPU kaudat-putamen, striatum bir parçası anlamına gelir, ve GP globus pallidus tanımlar. Her ikisi de, striatum hem de globus pallidus da bazal gangliyonlar olarak bilinen alt-kortikal çekirdeklerin bir gruba aittir. Otolog tam kan intrastriatal enjeksiyonu sonrası 24 saatte elde bregma bir koronal beyin bölümü 0.2mm anterior, (B) Temsilcisi fotomikrografıdır. <img alt="Şekil 2," src="/files/ftp_upload/4289/4289fig2.jpg" /> Şekil 2. Hematom histolojik tezahürüdür. Örnek hematoksilen ve eozin (H &amp; E), bir fare beyin lekeli koronal cryosection (10 mikron), bakteriyel kollajenaz intrastriatal enjeksiyonu (0.075 U) sonra 24 saatte hematom boyutu gösteren. LH = sol hemisfer, RH = sağ hemisfer. &lt;<html> img alt = &quot;Şekil 3&quot; src = &quot;/ files/ftp_upload/4289/4289fig3.jpg&quot; /&gt; Şekil 3,. Farelerde deneysel ICH takip nörofonksiyonel değerlendirmeler. Otolog kan (30 ul) veya bakteriyel kollajenaz intrastriatal enjeksiyonu (0.075 U) tekrarlanabilir nörofonksiyonel açıklarının neden oldu. (A) Deneysel İSH&#39;lı Fare ameliyat sonrası 24 ve 72 saatte sham operasyonu hayvanlara göre çok daha fazla sağa dönüşlerle gösterdi. Sol bacak (B) forepawın yerleştirerek kapasite ameliyattan sonra 24 ve 72 saatte İSH&#39;lı bozulmaktadır. Değerler ortalama ± SEM olarak ifade ve Öğrenci-Newman-Keuls yöntemi izledi rütbeler Varyans Kruskal-Wallis Tek Yönlü Analizi ile analiz edilmiştir. &lt;0.05 AP değeri istatistiksel olarak anlamlı kabul edildi; n = 6-12 grup başına, * P sham göre &lt;0.05. <a href="http://www.jove.com/files/ftp_upload/4289/4289fig3large.jpg" target="_blank">büyük bir rakam görmek için buraya tıklayın</a> . ŞEKIL 4 &quot;src =&quot; / files/ftp_upload/4289/4289fig4.jpg &quot;/&gt; Şekil 4. Farelerde deneysel ICH sonrası beyin su içeriğinin değerlendirilmesi. Otolog kan intraserebral enjeksiyonu (30 ul) veya önemli 24 (A) ipsilateral korteks ve bazal ganglionlar beyin su içeriğinin artış ve 72 saat yol bakteriyel kollajenaz (0.075 U) (B) ICH-indüksiyon sonrası. Değerler ortalama ± SEM olarak ifade ve Tukey post hoc testi ile takip Tek yönlü varyans analizi ile incelendi. &lt;0.05 AP değeri istatistiksel olarak anlamlı kabul edildi; n = 6-10, grup başına * P sham göre &lt;0.05. <a href="http://www.jove.com/files/ftp_upload/4289/4289fig4large.jpg" target="_blank">büyük bir rakam görmek için buraya tıklayın</a> .

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Çıkar çatışması ilan etti.

Intraserebral kanama (İSK) hayvan modelleri hastalığın patofizyolojisinin anlaşılmasına büyük katkı, ve genellikle bir preklinik ortamda yeni tedavi stratejileri geliştirmek ve değerlendirmek için kullanılır. Otolog kan veya bakteriyel kollajenaz intraparankimal enjeksiyonları kemirgenlerde ICH oluşturmak için köklü yöntem vardır. Her iki yöntem başlangıçta sıçan geliştirilmiştir, ancak transgenik ve nakavt suşların hızla artan durumu nedeniyle, fareler daha hemorajik beyin yaralanması ...

<table border="1"><tbody><tr><td> Malzeme </td><td> Şirket </td><td> Katalog Numarası </td><td> Yorumlamak </td></tr><tr><td> Stereotaksik Kafa Çerçeve </td><td> Stoelting Co </td><td> 51600 </td><td></td></tr><tr><td> Nanomit Şırınga Pompası </td><td> Harvard Apparatus </td><td> PY2 70-2217 </td><td></td></tr><tr><td> Hamilton Şırınga </td><td> Hamilton Company </td><td> 1725RN (250 ul) 1701 RN (10 ul) </td><td> 250 ul ve 10 ul şırıngalar için 26 Gauge iğne. </td></tr><tr><td> Mikrotur </td><td> Güzel Bilim Araçları </td><td> 18000-17 </td><td></td></tr><tr><td> Mikrotur çapak </td><td> Güzel Bilim Araçları </td><td> 19007-09 </td><td> 0.9 mm çapında </td></tr><tr><td> Kollajenaz Tip VII-S </td><td> Sigma-Aldrich </td><td> C2399 </td><td></td></tr><tr><td> Kapiler Tüpler Mikrohematokrit </td><td> Fisher Scientific </td><td> 22-362-574 </td><td> heparinize olmayan </td></tr><tr><td> Kemik Wax </td><td> Ethicon </td><td> W31 </td><td></td></tr><tr><td> Dikin </td><td> Ethicon </td><td> 1676G </td><td></td></tr><tr><td> Ketamin </td><td> Jhp İlaç </td><td> 42023-115-10 </td><td> Ketalar </td></tr><tr><td> Ksilazin </td><td> LLOYD Laboratuvarlar </td><td> 139-236 </td><td> AnaSed </td></tr></tbody></table>

modeling intracerebral hemorrhage in mice: injection of autologous blood or bacterial collagenase

Spontan intraserebral kanama (İSK) tüm inme ile ilişkili hastaneye yatışların% 10-15 ve hiçbir etkili tedavi 1,2 bugüne kadar mevcut olduğu için hesapları potansiyel olarak yaşamı tehdit eden nörolojik illet tanımlar. Çünkü insanlarda İSH heterojenliği, çeşitli preklinik modeller iyice 3 prospektif tedavi stratejileri keşfetmek için ihtiyaç vardır. Deneysel ICH yaygın otolog kan ya da bakteriyel ya da kollajenaz 4 arasında intraparenkimal enjeksiyon yoluyla kemirgenlerde meydana gelir. Uygun modeli kanama indüksiyon ve yaralanma ilerleme patofizyolojisi dayanarak seçilir. Kan enjeksiyon modeli bir hızla ilerleyen kanama taklit eder. Alternatif olarak, bakteriyel kollajenaz enzimatik genellikle birkaç saat içindeki 5 geliştikçe aktif kanama neden, beyin kılcal damarların bazal lamina bozar. Sonuç perihematomal ödem ve nöro açıkları fro ölçülebilirm modelleri hem. Bu çalışmada açıklanan ve otolog kan 6 modifiye edilmiş bir çift enjeksiyon modeli hem de bakteri kollajenaz 7&#39;nin bir ICH enjeksiyon modeli, her ikisi de hedef erkek CD-1 mice ve bazal gangliyonlar (corpus striatum). Değerlendirildi Biz ICH indüksiyon sonrası nörofonksiyonel açıkları ve 24. beyin ödemi ve 72 saat değerlendirilmiştir. Otolog kan (30 ul) veya bakteriyel kollajenaz (0.075U) arasında intrastriatal enjeksiyonu farelerde tekrarlanabilir nörofonksiyonel açıkları ve ameliyat sonrası anlamlı artış beynin 24 ödem ve 72 saat (p &lt;0.05) neden oldu. Sonuç olarak, her iki model de tutarlı hemorajik enfarkt verim ve preklinik ICH araştırma için temel yöntemlerini temsil eder.

Watch this Scientific Journal Video about Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase at JoVE.com

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Intraserebral kanama (İSK) ve Klinik ilgili hayvan modelleri hemorajik inme bilgimizi genişletmek ve yeni tedavi stratejileri incelemek gereklidir. Bu çalışmada, biz tanımlamak ve farelerin bazal ganglion (korpus striatum) içine otolog tam kan ya da bakteriyel kollajenaz ya tek taraflı enjeksiyon uygulamak iki ICH modelleri değerlendirir.

Farelerde Modelleme İntraserebral Kanama: Otolog Kan veya bakteriyel kollajenaz Enjeksiyon

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

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24.0K Görüntüleme.  Loma Linda University School of Medicine. Intraserebral kanama (İSK) ve Klinik ilgili hayvan modelleri hemorajik inme bilgimizi genişletmek ve yeni tedavi stratejileri incelemek gereklidir. Bu çalışmada, biz tanımlamak ve farelerin bazal ganglion (korpus striatum) içine otolog tam kan ya da bakteriyel kollajenaz ya tek taraflı enjeksiyon uygulamak iki ICH modelleri değerlendirir.

Video: Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Fare Modelleme İnme - Filament Model Orta Serebral Arter Tıkanıklığı

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

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Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral and cellular mucosal immune system1,2. 
 Microbiota is shielded from the immune system via a strong mucosal barrier. Infections and antibiotics are known to alter both the normal 
	gastrointestinal tract barrier and the composition of resident bacteria, which may result in possible immune abnormalities3.
HIV causes a breach in the gastrointestinal barrier with progressive failure of mucosal immunity and leakage into the systemic circulation of bacterial bioproducts, such as lipopolysaccharide and 
	bacterial DNA fragments, which contribute to systemic immune activation4-7. Microbial translocation is implicated in HIV/AIDS immunopathogenesis and response to therapy 4,8.
We aimed to characterise the composition of bacteria translocating in peripheral blood of HIV-infected patients. To pursue our aim we set up a PCR reaction for the panbacteric 16S ribosomial gene 
	followed by a sequencing analysis.
Briefly, whole blood from both HIV-infected and healthy subjects is used. Given that healthy individuals present normal intestinal homeostasis no translocation of microflora is expected in 
	these patients. Following whole blood collection by venipuncture and plasma separation, DNA is extracted from plasma and used to perform a broad range PCR reaction for the panbacteric 
	16S ribosomial gene9. Following PCR product purification, cloning and sequencing analyses are performed.

Our experiment will show how to perform a sequencing analysis of bacterial species translocating in peripheral blood of HIV positive patients.

Periferik Kan Bakteriyel Mikroflorası Sıralama: HIV ile enfekte olan hastalar ile bizim Deneyimi

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral ...

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

Fare intraserebroventriküler sayesinde Terapötik Ajanlar teslimi (ICV) ve intravenöz (IV) Enjeksiyon

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

Yerel ya da Sistemik Farmakolojik Müdahale sonrası Farelerin Uçucu Genel Anestezik Hassasiyet Değişiklikleri değerlendirilmesi

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective treatment and failure of large clinical trials aimed at hemostasis and clot removal demonstrate the need for further mechanism-driven investigation of ICH. This research may be performed through the framework provided by preclinical models. Two murine models in popular use include intrastriatal (basal ganglia) injection of either autologous whole blood or clostridial collagenase. Since, each model represents distinctly different pathophysiological features related to ICH, use of a particular model may be selected based on what aspect of the disease is to be studied. For example, autologous blood injection most accurately represents the brain&#39;s response to the presence of intraparenchymal blood, and may most closely replicate lobar hemorrhage. Clostridial collagenase injection most accurately represents the small vessel rupture and hematoma evolution characteristic of deep hemorrhages. Thus, each model results in different hematoma formation, neuroinflammatory response, cerebral edema development, and neurobehavioral outcomes. Robustness of a purported therapeutic intervention can be best assessed using both models. In this protocol, induction of ICH using both models, immediate post-operative demonstration of injury, and early post-operative care techniques are demonstrated. Both models result in reproducible injuries, hematoma volumes, and neurobehavioral deficits. Because of the heterogeneity of human ICH, multiple preclinical models are needed to thoroughly explore pathophysiologic mechanisms and test potential therapeutic strategies.

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

İntraserebral Kanama Fare Modellerinden olarak Otolog Blood veya Klostridyal Kollajenazının intrastriatal Enjeksiyon

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

Farelerde spontan metastatik renal hücreli karsinom (MRCC) modelleme ardından nefrektomi

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

Farelerde ultrason eşliğinde transtorasik intramiyokardiyal Enjeksiyon

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Farelerde Modelleme İnme: Distal Orta Serebral Arter Daimi Pıhtılaşma

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.