Funding for this work was provided by: AG007218 and NIH F32 AG031606.
Images were generated in the Core Optical Imaging Facility, which is supported by UTHSCSA, NIH-NCI P30 CA54174 (CTRC at UTHSCSA) and NIH-NIA P01AG19316.

Not: Tüm hayvan prosedürleri Teksas Sağlık Bilim Merkezi San Antonio Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından onaylanmış ve GELİYORUM kurallar ile uyumlu idi. 1. Kortikal Hazırlama anestezi <ol><li> Anestezi ikna etmek için oksijen ile karıştırılır% 2-3 isofluorane bir indüksiyon odasında fare yerleştirin. Fare kaynaklı olarak solunum hızı azalma gözlemleyin. Fare burun konisi taşınmaya hazır olup olmadığını belirlemek için fare pençe sıkıştırın. Not: Anestezi seviyesi herhangi in vivo hazırlanmasında önemli bir adımdır ve bakım küresel iskemi neden olacak bir düzeye ikna etmek değil alınmalıdır. </li><li> Fare yeterince uyuşturulduktan sonra, cerrahi / görüntüleme platformu hayvan transferi ve burun konisi farenin burnunu yerleştirin ve bir anestezi durumunu korumak için 1-1.2% isofluorane uygulayın. Fare sıcaklık co yatarken olduğundan emin olunntrolled ısıtma yastığı kalan prosedürlerin boyunca vücut ısısını (37 ° C +/- 0.5 ° C) muhafaza etmek. Anestezi altında iken kuruluğunu önlemek için gözleri üzerine veteriner merhem yerleştirin. </li><li> Sistemiyle birlikte sağlanan kuyruk veya ayak klibi kullanarak bir pulse oksimetre sistemini kullanarak fare fizyolojisini izleyin. Solunum hızı 50-65 nefesler / dk arasında muhafaza olup olmadığını kontrol edin. Hayvanın uzun vadeli hayatta kalmasını sağlamak için 97-98% arasında muhafaza edilir bpm ve oksijen doygunluğu 300-450 arasında kalp hızı kalmasını kontrol ediniz. </li><li> Fare yeterince uyuşturulduktan sonra, elektrikli makası kullanarak kafatası üzerinde saç tıraş etanol bez ardından kalan saç ve betadin temiz, kaldırın. Steril cerrahi ortamı sağlamak için üç kez bu prosedürü tekrarlayın. </li></ol> 2. Cerrahi Prosedür <ol><li> Tamamen temizlenmiş ve traşlı kafa derisi ile kranial fissu ortaya çıkarmak için fare kafa derisi 5 mm kesi yapmakres ve bregma bulmak için. </li><li> Kafatası örten kalan fasya kaldırmak için steril bir pamuk aplikatör kullanın. </li><li> Bregmadan stereotaksik koordinatlarını kullanarak parietal korteks örten kemik doku yapıştırıcı ile bir özel yapılmış paslanmaz çelik yüzük (Şekil 1) Tutkal: yanal -1 -3 mm ve: 2-4 mm. Not: tutkal, genellikle kemik üzerine halka yerleştirilmesinden sonra 2 dakika içinde ayarlar. </li><li> Fareyi stabilize etmek ve görüntüleme sırasında hareket etmesini önlemek için bir stereotaksik tutucu (Şekil 2) halkayı yapıştırın. </li><li> Cerrahi dereceli mikroskop altında yavaş yavaş bir hız kontrollü dremel benzeri aracını (Meisinger 3.9 mm matkap ucu) o delinmiş gibi alan seviyede tutmak için emin olun kullanarak kafatası içinde 1-2 mm&#39;lik bölümü matkap. Zigzag deseni kullanarak bunu başarmak. Isı önlemek için inşa, düşüğe matkap hızını ayarlamak ve sık sık ara verin. </li><li> Kranial kafatası görünüm tırmanmak olduğunda, inceltme devaminceltilmiş yüzey seviyede tutmak için aynı zig zag deseni kullanan bir neşter bıçak kullanarak kafatası kafatası ince tabakalar düzgün çıkarılmasını kolaylaştırmak için. Bir seferde kemik ince tabakalar kaldırmak için hafif bir baskı ile küçük doğrusal vuruş yapmak neşter bıçak ucunu kullanarak. Damar açıkça mikroskop aracılığıyla görülebilir oluncaya kadar bu devam edin. </li><li> Deneyci ile deler veya inceltme işlemi sırasında kafatası kırarsan, altta yatan korteks olası hasar hayvan euthanize. Not: Fare kafatası yaklaşık 300 um kalınlığında ve iki ince, kompakt kemik tabakaları (dış ve bir iç tabakası) ve kompakt kemik iki tabaka arasına sıkıştırılmış süngersi kemik tabakası oluşur. dış yoğun kemik tabakası ve süngersi kemiğin en geriye kalan küçük kemik yaklaşık 50 um tabaka içinde elde edilen 5 mm delik alanı içinde çıkarılır (Şekil 2B). Visualizdamarsal tirme son bozulmamış inceltilmiş kafatası yaklaşık 50 mikron kalınlığında olmasını sağlayacaktır. Bu kalınlığa inceltilerek zaman kafatası hala nedenle mevcut. </li></ol> 3. Mikroskop Set-up <ol><li> Objektif İnverter sahip bir ters mikroskop sistemi (konvansiyonel, konfokal veya iki foton sistemleri) kullanın. Not: Bu standart dik mikroskop kullanılması da mümkündür. sınırlayıcı faktör evre ve hedefleri arasındaki boşluk olacak. Sahneye Değişiklikler bu kurulum gerçekleştirmek için gerekli olabilir. </li><li> Kenara mikroskop tabanını bulunan bir ölçüye sahneye cerrahi / görüntüleme platformu sabitleyin. Not: Platform hedefi altında, cerrahi / görüntüleme platformu dikey hareketini sağlamak için bir laboratuvar jack kullanılarak yapılır. laboratuar jakı dört silindirik kutup yapıştırılmış bir plakaya monte edilir. (Bakınız Şekil 2). </li><li> 20x içeren objektif İnverter yerleştirinkranial pencere üzerinde objektif. Mikroskop okülerlerinde bakarak kranial pencere bulmak ve görüntüleme alanındaki hedefi konumlandırmak için harici bir ışık kaynağı kullanın. Not: Görüntüleme alanı damarsal varlığı ile ifade edilecektir. </li><li> Su bazlı hedefleri için, yapay beyin omurilik sıvısı (aCSF) kullanan orta vadesi potansiyeli (; 30 mM KCI; 130 mM NaCl 200 mM NaHCO 3,,, 30 mM HEPES ve 100 mM glukoz KH 2 PO 4 12 mM) görüntüleme sırasında kafatası boşluğuna kaçağı (Şekil 3). </li></ol> 4. Rose Bengal Boya hazırlanması, Yönetim ve İnme İndüksiyon <ol><li> Yapay beyin omurilik sıvısı (CSF), Rose Bengal, bir taze 20 mg / ml solüsyon hazırlamak; filtre ve uygulamadan önce sterilize edin. Yeniden ya da karışık edildikten sonra Gül Bengal tutmayın. Her bir deney için taze bir çözüm olun. </li><li> Rose Bengal s ise bir 0.1 mi kuyruk damarı enjeksiyonuBir 561 nm lazer ile kranial pencere konserve çözümün yeterli enjeksiyonu sağlamak. Not: Rose Bengal, beyin damar sisteminde, enjeksiyondan sonra 5 saniye içinde görselleştirilecektir. Tüm gemi Gül Bengal ile doldurulmalıdır. </li><li> Rose Bengal boya yeterli enjeksiyonunu takiben, belirli bir lezyon hacminin yeniden üretilebilirlik sağlamak için damar çapı (40-80 um) göre tromboz için uygun bir kap seçin. Kan akış yönü bakarak arterler ve venler arasında ayrım: arterler küçük çaplı damarları büyük çaplı hareket edecek, damarlar büyük çaplı damarları daha küçük hareket. Rose Bengal enjekte edildiğinde bu, kolayca görselleştirme ile gerçekleştirilir. </li><li> Aşağıdaki gibi mikroskop ayarını değiştirin: <ol><li> Bekleme süresini artırın. Not: Bu kullanılmaktadır mikroskop sistemine bağlı olarak değişir. </li><li> % 100 lazer gücünü artırın. </li><li> Kullanarak 1 kare / sn zaman dizisi görüntüleri toplayınMaksimum tarama hızı. </li></ol></li><li> Istikrarlı bir pıhtı damar içinde oluşana dek fareyi tarayın. Not: Bu genellikle sürekli tarama 5 dakika içinde ulaşılır (Bakınız Şekil 4). </li><li> Gül Bengal kullanarak pıhtı oluşumu indüksiyon sonrasında, geri mikroskop Görüntüleme alanından fareyi çıkarın. Dikkatle kranial kafatası paslanmaz çelik halkasını çıkartın. Herhangi bir kanama kranial pencereyi inceleyin. Kanama oluşursa, deney sonlandırın. </li><li> Kafatası üzerinde insizyon kapatmak için 6.0 monofilament sütür kullanın. Enfeksiyonu önlemek için dikiş hattı boyunca antibiyotik merhem yerleştirin. Buprenex (0.05 mg / kg), ağrı yönetimi için üç gün boyunca deri altından her 12 saatte enjekte edilir. </li><li> Tamamen uyanık ve serbestçe hareket kadar anestezi çıkarıldıktan sonra bir kurtarma odasına fare dönün. </li><li> Daha sonra daha fazla araştırma için temiz bir kafes fare dönün. </li></ol> 5. Daha sonraki Günlerinde Boyuna Görüntüleme <ol><li> Sonraki gün sonrası Fototromboz boyuna görüntüleme gerçekleştirmek için aşağıdaki yöntemleri kullanır. <ol><li> Yöntemlerin Bölüm 1&#39;de açıklandığı gibi fare anestezisi. </li><li> Yeterli anestezi sırasında önceden delinmiş görüntüleme alanını örten cilt yeniden açmak için herhangi bir kalıntı sütür kaldırarak saç derisini yeniden açın. </li><li> Kafatası örten kalan fasya kaldırmak için steril bir pamuk aplikatör kullanın. </li><li> Önceki görüntüleme alanını örten kemik doku yapıştırıcı ile bir özel yapılmış paslanmaz çelik yüzük (Şekil 1) Tutkal. </li><li> Fareyi stabilize etmek ve görüntüleme sırasında hareket etmesini önlemek için bir stereotaksik tutucu (Şekil 2) halkayı yapıştırın. </li><li> Daha önce inceltilmiş kafatası altında yatan damar bulun. Daha önce neden pıhtı varlığını doğrulamak için, FITC-dekstran kuyruk damarı enjeksiyonu kullanın. </li><li> In kapatmak için 6.0 monofilament sütür kullanınKafatasının üzerinde eksizyondur. Enfeksiyonu önlemek için dikiş hattı boyunca antibiyotik merhem yerleştirin. Buprenex (0.05 mg / kg), ağrı yönetimi için üç gün boyunca deri altından her 12 saat enjekte edilir. </li><li> Tamamen uyanık ve serbestçe hareket kadar anestezi çıkarıldıktan sonra bir kurtarma odasına fare dönün. </li></ol></li></ol> İnme indüksiyon 6. Doğrulama (Post-mortem) <ol><li> Bir çalışmanın bitiminde, Şekil 5&#39;te gösterildiği gibi 2,3,5-trifeniltetrazolyum klorür (TTC) boyama kullanarak strok hacmini doğrulayın. Tam bir yöntem 12&#39;de bulunabilir. </li></ol>

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Yazarlar hiçbir rakip mali çıkarlarını olduğunu beyan ederim.

İnsan uygulamaya hayvandan deneysel inme patofizyoloji çevirmek için yetenek yetersizliği ile rahatsız olmuştur. Ancak, bu tür fototromboz modeli olarak hayvan modellerinde, kullanımı gelişmiş inme patofizyolojisi anlayış ve inme sonrası nöro sağlamak için yeni tedavi yaklaşımları keşif için izin verir. Küçük kortikal vuruş ve Fototrombotik modeli tarafından üretilen microinfarctions yüksek oranda görüldüğü ve Amerika Birleşik Devletleri nüfusunun (yaklaşık 11 milyon kişi) her yıl...

Hemen Gül Bengal indüksiyon aşağıdaki vivo hücresel yanıtları tekniği tarif izin görselleştirme sağlam bir fare Fototromboz. Rose Bengal (4,5,6,7-tetrakloro-2 &#39;, 4&#39;, 5 &#39;, 7&#39;-tetraiodofluorescein) hayvan modellerinde, iskemik inme (fare ve sıçan) ikna etmek için kullanılan ışığa boyadır. Bir 564 nm lazer ışığı ile inceltilmiş kafatası yoluyla kuyruk damarından ve daha sonra aydınlatma aracılığıyla RB bir bolus enjeksiyonunu takiben, trombüs fizyolojik inme 1 neden indüklenir. yöntem aslen 1977 yılında Rosenblum ve El-Sabban&#39;ın tarafından tarif edilmiştir ve daha sonra orta 1980&#39;ler 1,2 Watson tarafından uyarlanmıştır. Kısaca, Rose Bengal, daha sonra, doku faktörü, pıhtılaşma kaskadının bir başlatıcı aktive reaktif oksijen türlerinin üretimi üretir yeşil uyarma ışık (bizim örneğimizde 561 nm lazer) ile ışınlanır. koagülasyon kaskad indüksiyon les iskemik üretirKlinik inme 3 patolojik alakalı iyon. İnme nöronlar, glia, endotel ve bağışıklık sistemi de dahil olmak üzere birçok farklı hücre tiplerinin etkileşimi nedeniyle karmaşık bir patofizyolojisi vardır. En iyi tekniği seçimi, belirli bir hücresel süreç birden hususlar gerektirir incelemek. Deneysel teknikler üç kategoriden birine genel olarak düşmek: in vitro, her biri avantaj ve dezavantajları ile in vivo ve siliko in vitro çalışmalar, doğal ortamdan hücrelerin çıkarılması birincil dezavantajı vardır ve bu nedenle sağlam bir görülen etkileri yeniden olmayabilir. hayvan yaşam. in vivo teknikler artmış öteleme öneme sahip hastalık durumlarının geliştirilmiş deneysel çoğaltma sağlamak. silico olarak genellikle bir hastalık veya hücresel sürecin bilgisayar modelleme anlamına gelir ve giderek sınava potansiyel ilaç etkileşimleri incelemek için kullanılan süreple, toplanan herhangi bir bilgi hala hücreleri veya canlı doku test edilmelidir. laboratuar ortamında inme ideal bir model insan popülasyonunda görülen benzer patolojik özellikleri göstermelidir. Insan nüfusunun inme ortak fizyolojik özellikleri varken, deneyimli yaralanma türüne bağlı olarak pek çok farklılıklar vardır. Insan nüfusunun İnme çeşitli enfarktüs hacimleri yanı sıra her patoloji ile ilgili mekanizmalar farklılıklara neden olarak küçük veya büyük damar oklüzyonlarını, hemorajik lezyonlar ve arter veya kardiyo-embolisine arter-oluşur. Hayvan inme modelleri kullanan avantajı, insan inme özelliklerini taklit eden tekrarlanabilir infarktlar üretilmesidir. orta serebral arter tıkanıklığı (emboli veya endovasküler filament yöntemleri) hangi modeller uzak MCAO ve fototromboz modeli: En yaygın hayvan inme modelleri kullanarak arter tıkanıklığı sayılabilir. Avantajları BirHer modelin d dezavantajları (4 ve 5) başka bir yerde gözden geçirilmiştir. Küresel iskemik modelleri (MCAO), gerçekleştirmek için nispeten kolay odak inme modelleri daha insan inme daha az alakalı iken. Buna ek olarak, bu yöntemler, yeniden üretilebilir, beyin enfarktüsü lezyonlara neden olan oldukça değişkendir. deneyci MCAO modeller üzerinde açık bir avantaj sağlayarak, onların deneylerini kontrol olarak fototromboz modeli uzun gibi son derece tekrarlanabilir. Bununla birlikte, mikrovasküler hasara modeli asgari penumbra, hücre 6,7 kurtarılabilen olduğu düşünülmektedir alan görüntülemek için tarif edilmiştir. Buna ek olarak, vazojenik ödem ve sitotoksik ödem oluşumu, aynı zamanda görüntüleme alanında aşağıdaki ışınlama indüklenebilir. Bu kısıtlamalara rağmen tekniği inme 8, 9, 10, 11 aşağıdaki birçok fizyolojik süreçlere yeni bir bakış açısı sağlamıştır.

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rose bengal photothrombosis by confocal optical imaging in vivo: a model of single vessel stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Bu yöntemin amacı, bir 561 nm lazer ışığı ile inceltilerek kafatası kuyruk damarından ve daha sonra aydınlatma aracılığıyla RB bir bolus enjeksiyonu takiben hayvan modellerinde, bir iskemik inme (fare ve sıçan) neden oldu. Şekil 4 &#39;de resim olarak 0, 1, 1.5 ve 2 dk&#39;da alanının ışınlanması sonra tek bir tekne içinde pıhtı oluşumunun ilerlemesini göstermektedir. Pıhtı oluşumu öncesinde, tüm geminin Rose Bengal serbest akan nedeniyle beyazdır. Kabın ışınlama in...

Watch this Scientific Journal Video about Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke at JoVE.com

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Konfokal Optik Görüntüleme ile Rose Bengal Fototromboz<em&gt; In Vivo</em&gt;: Tek Gemi İnme Bir Model

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

rose-bengal-photothrombosis-confocal-optical-imaging-vivo-model

Research

JoVE Journal

Medicine

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

14.9K Görüntüleme.  The University of Texas Health Science Center San Antonio. Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection. 

Video: Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Bebeklerde Serebral Metabolizma ve Hemodinamiğin Noninvaziv Optik Ölçüm

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Tıp

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Flüoresans Konfokal videomikroskopi kullanılarak tümör Angiogenesis İn vivo

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Subaraknoid Kanama bir fare modeli

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

Floresan SapC-DOPS Nanovesicles kullanma Beyin Tümörleri ve Artrit Vivo Optik Görüntüleme

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Bir olarak Beyin Microslices arasında excitotoxic uyarılması<em&gt; In vitro</em&gt; İnme Model

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Kombine<em&gt; In vivo</em&gt; Optik ve uCT Görüntüleme Farelerde bir Ortopedik İmplant Enfeksiyon Enfeksiyon, Enflamasyon ve Kemik Anatomisi Monitör

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Kemoterapi kaynaklı Vasküler Toksisite - Gerçek zamanlı<em&gt; In vivo</em&gt; Gemi Düşüklüğü Görüntüleme

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Farelerde Omurilik Yaralanma Modeli Olarak Odak iskemisi Fototromboz kaynaklı

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Bir Fulminan Miyokardit Modelinde Işık Yeri Mikroskopisi İle Lökosit İnfiltrasyonunun Nicel Görselleştirilmesi

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

İnsan vücudunun fizyolojisini bütünleştiren 4 damarlı Örnekleme Yaklaşımı<emIn vivo</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...