Yazarlar nazik gibi bu araştırmayı yürütmek için fon Alerji ve Bulaşıcı Hastalığı için Ulusal Enstitüsü&#39;ne teşekkür ederim.

Dozlandırıcılara Bileşenleri 1. Hazırlık <ol><li> delinerek veya sadece bileme veya taşlama çarkının veya makas keskin bir çifti ile alt keserek 0.5 ml polipropilen mikrosantrifüj tüpü alt delikli. Delik merkezindedir ve çapı en fazla 2 mm ve çapı en az 1 mm (Şekil 1) olduğundan emin olun. </li><li> mikrosantrifüj tüpü üst delikli. Bir 22. Matkap ucu (çapı yaklaşık olarak 4 mm) kullanılarak, kapağın merkezi içinde bir delik açın. Bu kayma ucu şırınga (Şekil 1) uyacak bir delik ile sonuçlanır. </li><li> Hazırlama ince ızgaralar mikrosantrifüj tüplerine sokulacak. . 304 paslanmaz çelik tel kumaş, 0.0045&quot; tel çapı olan 60 x 60 mesh ekran yapmak makaslarına ekranları kesme ya da 5 mm (Şekil 2), bir dış çapına bir kalıp kullanılarak yumruk. </li></ol> Dozlandırıcılara 2. Montaj <ol><li> ya forseps veya kör uç iğne ucunu kullanarak, bir mikrosantrifüj tüpü içine ekran takıp açıklığı kapatacak şekilde alt içinde koltuk. Bu işletilmesinden önce toz desteklemeye hizmet edecektir. </li><li> Basın bir kör uç iğnenin ucuna mikrosantrifüj ucunun dar ucuna sığacak. elle veya bir tezgah üstü basın ve rehber eşliğinde yapın. Yavaşça sıkı esnek bir uyum sağlamak için parçaları temizlemek için etanol ile tüpün iğne ve alt üst silin. </li></ol> 3. dozlandırıcılara Dolum <ol><li> bir analitik mikro terazi kullanılarak, cihaz dara ve dozörü içine tozunun istenen miktarının yükleyin. dispersiyon için tipik bir toz kütlesi 1 ila 10 mg arasında değişir. </li><li> Bir kez dolu, pamuk bir ifraz ve forseps bir çift mikrosantrifüj tüpü üst takın. Bu şırınga içine çekme veya tozun dağılması için dozörü hava akışını izin verirken, düşmesini toz önleyecektir. </li><li&gt; Mikrosantrifüj tüpü kapağını kapatın. </li><li> hemen kullanılan değilse, ortam nemi için, toz maruz kalmasını en aza indirmek için laboratuar film bir miktar ile üst mühür. Not: Genellikle, önceden yüklenmiş dozörler aktif farmasötik bileşen (API) depolama gereksinimlerine göre saklanmalıdır. Genellikle, aerosol tozlar neme karşı duyarlı olan ve kurutulmuş saklanmalıdır. </li></ol> 4. Aygıt Tetikleme <ol><li> Uygulamaya özel olarak arzu edilen hacim, şırıngayı geri çekin. gine domuzlarında intrapulmoner uygulama için, 2 mL kullanın. Dispersiyon etkinlik seviyesi ve çalıştırma sırasında iletilen havanın hızı, aynı zamanda tozun 7 doğasında dağılım özellikleri ile ilgilidir. dozörü üst delik kayma ucu şırınga barındırmaktadır. Bir Luer-kilit şırınga bir adaptör ile kullanılabilmektedir. </li><li> dozörü arka ucunu şırınga yerleştirin. </li><li> t içine dozörü iğne ucunuMaruziyet bölmesi bağlantı noktası dozaj. Cihazın üzerinden doğrudan hayvana veya poz bölmesi veya analitik araç (Şekil 3) içine tozun dışarı atılması, zorla şırınga basın. </li></ol>

<ol>
	<li>Young, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhaled+Pyrazinoic+Acid+Esters+for+the+Treatment+of+Tuberculosis.">Inhaled Pyrazinoic Acid Esters for the Treatment of Tuberculosis.</a> Pharmaceutical Research. , 1-11 (2016).</li><li>Hinds, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aerosol Technology: Properties, Behavior, and Measurement of Airborne Particles. , (2012).</li><li>Islam, N., Gladki, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+powder+inhalers+(DPIs)-A+review+of+device+reliability+and+innovation.">Dry powder inhalers (DPIs)-A review of device reliability and innovation.</a> International Journal of Pharmaceutics. 360 (1-2), 1-11 (2008).</li><li>Chan, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+powder+aerosol+delivery+systems:+current+and+future+research+directions.">Dry powder aerosol delivery systems: current and future research directions.</a> J Aerosol Med. 19 (1), 21-27 (2006).</li><li>Vehring, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmaceutical+Particle+Engineering+via+Spray+Drying.">Pharmaceutical Particle Engineering via Spray Drying.</a> Pharmaceutical Research. 25 (5), 999-1022 (2008).</li><li>Bosquillon, C., Lombry, C., Pr&#233;at, V., Vanbever, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+formulation+excipients+and+physical+characteristics+of+inhalation+dry+powders+on+their+aerosolization+performance.">Influence of formulation excipients and physical characteristics of inhalation dry powders on their aerosolization performance.</a> Journal of Controlled Release. 70 (3), 329-339 (2001).</li><li>Hickey, A. J., Concessio, N. M., Van Oort, M. M., Platz, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+the+dispersion+of+dry+powders+as+aerosols.">Factors influencing the dispersion of dry powders as aerosols.</a> Pharmaceutical Technology. 18 (8), 58-64 (1994).</li><li>Garcia-Contreras, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhaled+large+porous+particles+of+capreomycin+for+treatment+of+tuberculosis+in+a+guinea+pig+model.">Inhaled large porous particles of capreomycin for treatment of tuberculosis in a guinea pig model.</a> Antimicrob Agents Chemother. 51 (8), 2830-2836 (2007).</li><li>Durham, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spray+Dried+Aerosol+Particles+of+Pyrazinoic+Acid+Salts+for+Tuberculosis+Therapy.">Spray Dried Aerosol Particles of Pyrazinoic Acid Salts for Tuberculosis Therapy.</a> Molecular Pharmaceutics. 12 (8), 2574-2581 (2015).</li><li>Sung, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+powder+nitroimidazopyran+antibiotic+PA-824+aerosol+for+inhalation.">Dry powder nitroimidazopyran antibiotic PA-824 aerosol for inhalation.</a> Antimicrob Agents Chemother. 53 (4), 1338-1343 (2009).</li><li>Guillon, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+delivery+of+dry+powders+to+rats:+tolerability+limits+of+an+intra-tracheal+administration+model.">Pulmonary delivery of dry powders to rats: tolerability limits of an intra-tracheal administration model.</a> Int J Pharm. 434 (1-2), 481-487 (2012).</li><li>Morello, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry-powder+pulmonary+insufflation+in+the+mouse+for+application+to+vaccine+or+drug+studies.">Dry-powder pulmonary insufflation in the mouse for application to vaccine or drug studies.</a> Tuberculosis. 89 (5), 371-377 (2009).</li></ol>

Portions of the pharmacokinetic data was presented at Respiratory Drug Delivery 2014 in Fajardo, Puerto Rico and dosator depictions and additional pharmacokinetic data were presented at the annual meeting of the American Association of Pharmaceutical Scientists 2015 in Orlando, Florida.

Hayvanlar birincil parçacık boyutuna dağılmış bir aerosol büyük bir bölümünü gösteren, pasif sadece burundan solunum yoluyla terapötik dozunu almak için dozlandırıcılara dispersiyon uygundu. Bu dozörler analitik veya etkili deneyler için in vitro toz performansının değerlendirilmesi ve genel amaçlı kuru toz aerosol dispersiyon içinde, endotrakeal uygulama için kullanılabilir. intrapulmoner teslimat için kullanılan, doku hasarına neden olabilir ve farmakokinetik sonuçlar?...

<html> Yeni terapötik ürünlerin geliştirilmesi bir hayvan modelinde etkinlik testleri gerektirir. Uygulama pulmoner yol yerel olarak ve sistematik olarak 1 ilaç vermek için kullanılabilir. Kuru toz aerosoller değerlendirilmesi bir maruziyet hazne içinde veya direkt endotrakeal uygulama için yüksek konsantrasyonlarını korumak için etkili bir dağılım mekanizması gerektirir. Çözeltiler, toz aerosoller kuru pasif soluma ile hayvanları ortaya çıkarmak için mevcut olmakla birlikte, en fazla 2, verilen dozun büyük bir fazlalıkta toz kitleleri gerektirir. Bu araştırma veya erken gelişim aşaması geleneksel aerosol dağıtım sistemleri için doz dağıtım gereksinimlerini desteklemek üzere yetersiz ilaç kullanılabilir gibi erken fizibilite çalışmaları yürütüyor engellemektedir. bir aerosol ilaç ürününü tasarlarken, aerodinamik performansı temin verimi ve etkinliği, doğrudan ilgili olabilir&gt; 3. Bir aerosol haline toz dispersiyon parçacık arası kuvvetlerinin üstesinden gelmek için yeterli bir enerji girişini gerektirir ve parçacık mühendisliği yaklaşımları partikül büyüklüğü, aerosol performansı 4, 5, 6 artırabilir. Bu analitik amaçlar için bir maruziyet sistemi veya kuşak içine doğrudan pulmoner insüflasyon, dispersiyon amacıyla etkili bir mühendislik yardımıyla kuru toz aerosol aerosol haline bir dispersiyonunun (dozator) geliştirdik.

<table border="0" cellpadding="0" cellspacing="0" width="1158">
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			<td height="21" style="height:21px;width:695px;">0.5 mL microcentrifuge tube</td>
			<td style="width:139px;">VWR</td>
			<td style="width:119px;">89000-026</td>
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			<td height="23" style="height:23px;">High-Volume Particle-Filtering Stainless Steel Wire Cloth, Woven, 304 Stainless Steel, 60 x 60 Mesh, .0045&#34; Wire Diameter</td>
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			<td height="21" style="height:21px;width:695px;">Cotton balls</td>
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			<td style="width:119px;">54845T16</td>
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			<td height="21" style="height:21px;width:695px;">Parafilm M&#174; Laboratory Film</td>
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disposable dosators for pulmonary insufflation of therapeutic agents to small animals

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver drugs locally and systemically. Evaluation of dry powder aerosols necessitates an efficient dispersion mechanism to maintain high concentrations in an exposure chamber or for direct endotracheal administration. While solutions exist to expose animals by passive inhalation to dry powder aerosols, most require masses of powder in large excess of the dose delivered. This precludes conducting early feasibility studies as insufficient drug is available at the research or early development stage to support the dose delivery requirements for conventional aerosol delivery systems. When designing an aerosol drug product, aerodynamic performance can relate directly to delivery efficiency and efficacy. Dispersion of powder into an aerosol requires energy input sufficient to overcome interparticulate forces, and particle engineering approaches can substantially improve aerosol performance. We have developed a dispersion system (dosator) which can aerosolize engineered dry powder aerosols efficiently for the purpose of direct pulmonary insufflation, dispersion into an exposure system or generation for analytical purposes.

Gibi kolaylıkla dağılabilen tozlar için püskürtülerek kurutulmuş pulmoner yoldan 5, 8, 9, hedefi, dozörler cihazın dışında bir bolus dozu veren tek olanlar. Pasif inhalasyon sistemleri için in vitro parçacık karakterizasyonu, canlı hayvanlarda doğrudan intrapulmoner uygulama ve aerosol üretimi de dahil olmak dozlandırıcılara için bir çok uygulama vardır...

Watch this Scientific Journal Video about Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals at JoVE.com

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals

Pulmoner dağıtım için ilaçların geliştirilmesi sırasında, bir hayvan modelinde, farmakokinetik ve etkinliğini değerlendirmek için gereklidir. Biz kemirgenlere intrapulmoner kuru toz, aerosol yönetmek için kullanılabilir of-the-shelf bileşenlerden tek kullanımlık bir aerosol dağılım sistemi oluşturmak için bir yöntem sunulmaktadır.

Küçük Hayvanlarda Terapötik Ajanların Pulmoner Burundan çekmek için tek kullanımlık dozlandırıcılara

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver ...

disposable-dosators-for-pulmonary-insufflation-therapeutic-agents-to

Research

JoVE Journal

Medicine

7.6K Görüntüleme.  RTI International.  Pulmoner dağıtım için ilaçların geliştirilmesi sırasında, bir hayvan modelinde, farmakokinetik ve etkinliğini değerlendirmek için gereklidir. Biz kemirgenlere intrapulmoner kuru toz, aerosol yönetmek için kullanılabilir of-the-shelf bileşenlerden tek kullanımlık bir aerosol dağılım sistemi oluşturmak için bir yöntem sunulmaktadır. 

Video: Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.

Bronş Thermoplasty: Şiddetli Astım Yeni Bir Tedavi Yaklaşımı

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

Tıp

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

İnsan Takip edilen-Lusiferaz Malign Periferik Sinir Kılıf Tümör Hücreleri Ortotopik Xenografting In vivo</emAday Terapötik Ajanlar> Test

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

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 ...

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Farelerde Uzun Süre Whole Body Hipertermi için protokol

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

Kanser BİYOMARKÖRLERİN Yerelleştirme Küçük Hayvanlarda Bilgisayarlı Tomografi eşliğinde Zaman alanı Diffüz Floresans Tomografi

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

Sigara Duman maruz kalan Fare Akciğer Amfizem ve Küçük Havayolu Yaptırma otomatik ölçüm

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3&#8242; processing endoribonuclease, tRNase ZL, to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.

Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

DOĞRU gen susturma: Potansiyel Kanser Tedavi Ajanlar için heptamer tipi Küçük Rehberi RNA Kütüphanesi Taranması

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Amiloid-β ve Tau Nöropatolojisinin Transgenik Fare Modellerinde Spontan Dalgalanmanın Değerlendirilmesi, Yeni Nesne Tanıma ve Limb Klapması

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ultimately lead to new knowledge concerning the causes of or new ways to inhibit or treat plasma extravasation. It is important that researchers have the proper tools, including the best methods available, for studying plasma extravasation. In this article, we describe a protocol, using the Evans blue dye method, for assessing plasma extravasation in the organs of FVBN mice. This protocol is intentionally simple, to as great a degree as possible, but provides high quality data. Evans blue dye has been chosen primarily because it is easy for the average laboratory to use. We have used this protocol to provide evidence and support for the hypothesis that the enzyme neprilysin may protect the vasculature against plasma extravasation. However, this protocol may be experimentally used and easily adapted for use in other strains of mice or in other species, in many different organs or tissues, for studies which may involve other factors that are important in understanding, preventing, or treating plasma extravasation. This protocol has been extensively optimized and modified from existing protocols, and combines reliability, ease of use, economy, and general availability of materials and equipment, making this protocol superior for the average laboratory to use in quantifying plasma extravasation from organs.

In this article, an economical, optimized, and simple protocol is described which uses the Evans blue dye method for assessing plasma extravasation in the organs of FVBN mice that can be adapted for use in other strains, species, and other organs or tissues.

En iyi duruma getirilmiş bir Evans mavi Mouse vasküler sızıntı değerlendirmek için iletişim kuralı

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Yetişkin sıçan böbrek üzerinden olduğu gibi Glomeruli yalıtmak için verimli bir eleme yöntemi

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...