저자에 친절하게 좋아하는이 연구를 수행 할 수있는 자금에 대한 알레르기 및 전염병 국립 연구소 감사합니다.

Dosators의 부품의 제조 1 <ol><li> 뚫어하거나 또는 연삭 숫돌이나 위의 날카로운 한 쌍의 하단을 절단하여 0.5 ㎖의 폴리 프로필렌 미세 원심 튜브의 바닥을 관통. 구멍이 중앙에 직경하게는 2 mm 이상의 직경보다 작지 1 mm (도 1)인지 확인. </li><li> microcentrifuge 관의 상단을 관통. A # 22 드릴 비트 (직경이 약 4mm)를 사용하여 캡의 중앙에 구멍을 드릴. 이것은 슬립 주사기 팁 (도 1)에 맞는 구멍을 초래한다. </li><li> 준비 미세한 스크린은 마이크로 원심 튜브에 삽입한다. . 304 스테인리스 철망 &quot;0.0045 선 직경 60 X 60 메쉬의 스크린을 주석 자르는으로 화면 컷 또는 5mm (도 2)의 외경에 다이를 사용하여, 펀치. </li></ol> Dosators 2. 총회 <ol><li> 어느 겸자 무딘 팁 바늘의 끝을 사용하여, 마이크로 원심 튜브에 화면을 삽입하고 개구부를 덮도록 바닥에 안착. 이 작동하기 전에 분말을 지원하는 역할을합니다. </li><li> 눌러 뭉툭한 팁 바늘의 끝 부분에 마이크로 원심 끝의 좁은 끝을 맞습니다. 손으로 또는 벤치 탑 프레스 및 가이드와 함께이 작업을 수행합니다. 부드럽게 꽉 탄력 적합을 보장하기 위해 부품을 청소하는 에탄올로 튜브의 바늘과 하단의 상단을 닦으십시오. </li></ol> 3. Dosators를 작성 <ol><li> 분석 마이크로 저울을 사용하여 용기를 장치하고 dosator 분말에 소 정량의로드. 분산의 일반적인 분말 덩어리는 1 내지 10 mg의 범위이다. </li><li> 일단 채워진면의 분담 및 한 쌍의 집게로 microcentrifuge 관의 상단 플러그. 이 주사기에 그리거나 분말을 분산하기 위해 dosator로 공기 흐름을 허용하면서, 밖으로 떨어지는 분말을 방지 할 수 있습니다. </li><l난&gt;은 microcentrifuge 관 뚜껑을 닫습니다. </li><li> 즉시 사용하지 않는 경우, 주변 습도에 분말의 노출을 최소화하도록 랩 필름 소량의 상단을 밀봉. 주 : 일반적으로, 프리로드 dosators 활성 제약 성분 (API)의 저장 요구에 따라 저장한다. 종종 에어로졸 분말은 민감한 습기하고 건조 된 보관해야합니다. </li></ol> 4. 장치 작동 <ol><li> 응용 프로그램의 특정 될 수있다 원하는 볼륨에 주사기를 다시 그립니다. 기니 돼지 폐내 투여를 위해,이 용액을 사용한다. 분산 효율은 볼륨 및 작동시 전달되는 공기의 속도뿐만 아니라, 분체 (7)의 고유 산성 관련된다. dosator의 상단 구멍 슬립 팁 주사기를 수용한다. 루어 락 주사기는 어댑터와 함께 사용할 수있다. </li><li> dosator의 백 엔드에 주사기를 삽입합니다. </li><li> t에 dosator의 바늘 끝을 삽입그는 노출 챔버에 포트를 투여. 장치로부터 직접 또는 동물에 노출 챔버 또는 분석 기기 (도 3)로 분말을 추방 강제 주사기 우울. </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.

동물 차 입자 크기로 분산 된 에어로졸의 큰 비율을 나타내는 수동적 코 호흡만을 통해 치료 용량을 수신위한 dosators로부터 분산액이 적합했다. 이러한 분석은 dosators 또는 효능 실험 관내 분말 성능 평가, 다목적 건조 분말 에어로졸 분산, 기관 내 투여 용으로 사용될 수있다. 폐내 전달을 위해 사용되는 경우, 조직 손상의 원인 및 약동학 적 결과를 왜곡 할 수 이상 - 인플레이션 폐의...

<html> 새로운 치료제의 개발은 동물 모델에서 효능 테스트를 필요로한다. 폐 투여 경로는 국소 적 및 전신적 1 약물을 전달하기 위해 이용 될 수있다. 건조 분말 에어로졸 평가 노광 챔버에 직접 또는 기관 내 투여 용으로 높은 농도를 유지하는 효율적인 분산 메커니즘을 필요로한다. 솔루션은 분말 에어로졸을 건조 수동 흡입에 의해 동물을 노출 존재하지만, 대부분은 2 전달 용량의 과량으로 분말의 질량을 필요로한다. 이 연구 또는 초기 개발 단계는 기존의 에어로졸 전달 시스템의 용량 전송 요구 사항을 지원하기에 불충분 한 약물을 사용할 수 조기 타당성 조사를 실시 배제. 에어로졸 제제를 설계 할 때, 공기 역학적 성능을 전달 효율과 효능에 직접 관련 될 수&gt; 3. 에어로졸 분말로 분산 interparticulate 힘을 극복하기에 충분한 에너지 입력을 필요로하고, 입자 공학적 접근 실질적 에어로졸 성능 4, 5, 6을 향상시킬 수있다. 우리의 분석을 위해 노광 시스템이나 폐 생성에 직접 취입, 분산 목적으로 효율적으로 설계된 건조 분말 에어로졸을 에어로졸 화 할 수있는 분산 시스템 (dosator)를 개발 하였다.

<table border="0" cellpadding="0" cellspacing="0" width="1158">
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	<tbody>
		<tr height="21">
			<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>
			<td style="width:207px;"></td>
		</tr>
		<tr height="23">
			<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>
			<td style="width:139px;">McMaster Carr</td>
			<td style="width:119px;">9230T44</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless Steel Dispensing Needle, Straight, 18 Gauge, 1&#34; Long</td>
			<td style="width:139px;">McMaster Carr</td>
			<td style="width:119px;">75165A676</td>
			<td>Any luer-fit needle will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Cotton balls</td>
			<td style="width:139px;">McMaster Carr</td>
			<td style="width:119px;">54845T16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Parafilm M&#174; Laboratory Film</td>
			<td style="width:139px;">VWR</td>
			<td>100229-550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Black-Oxide High-Speed Steel Jobbers&#39; Drill Bit, Wire Gauge 22, 3-1/8&#34; Overall Length, 1.8&#34; Drill Depth, 135Deg Point</td>
			<td style="width:139px;">McMaster Carr</td>
			<td style="width:119px;">2901A195</td>
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		</tr>
	</tbody>
</table>

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.

같은 쉽게 산성 분말 용 분무 건조 된 폐 전달 5, 8, 9 의도에서, dosators 장치로부터 일시 투여 전달 이들. 수동 흡입 시스템을위한 생체 입자 특성, 살아있는 동물에서 직접 폐내 관리 및 에어로졸 세대를 포함하여 dosators에 대한 많은 응용 프로그램이 있습니다. <p class="jove_content" fo:keep-to...

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

폐 전달을위한 약물의 개발 과정은 동물 모델에서 약물 동력학과 효능을 평가하는 것이 필요하다. 우리는 설치류 폐내 건조 분말 에어로졸을 투여하는데 사용될 수의 상용 성분으로부터 일회용 에어로졸 분산 시스템을 구축하는 방법을 제시한다.

작은 동물에 대한 치료제의 폐 통기를위한 일회용 Dosators

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 조회수.  RTI International.  폐 전달을위한 약물의 개발 과정은 동물 모델에서 약물 동력학과 효능을 평가하는 것이 필요하다. 우리는 설치류 폐내 건조 분말 에어로졸을 투여하는데 사용될 수의 상용 성분으로부터 일회용 에어로졸 분산 시스템을 구축하는 방법을 제시한다. 

비디오: 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.

기관지 Thermoplasty : 심한 천식에 소설 치료 접근

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

연구

의학

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.

대한 인간 루시페라제 태그가 추가된 악성 주변 신경 쉬스 종양 세포 Orthotopic Xenografting 생체내에</em후보 치료 대리점> 테스트

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.

마우스에 Intracerebroventricular을 통해 치료 대리점 납품 (ICV)과 정맥 (IV) 사출

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.

마우스의 긴 기간 온 몸이 고열에 대한 프로토콜

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.

암 생체 현지화위한 작은 동물의 계산된 Tomography 주 도형 시간 도메인 미만성 형광 Tomography

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.

담배 연기에 노출 된 마우스의 폐 폐기종 및 작은기도 리모델링 자동 측정

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.

TRUE 유전자 음소거 : 잠재적 인 암 치료제에 대한 헵 타머 형 소형 가이드 RNA 라이브러리의 심사

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.

아밀로이드 -β 및 타우 신경 병증의 유전자 변형 마우스 모델에서 자발적 교대, 새로운 대상 인식 및 사지 걸림의 평가

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.

최적화 된 에반스 블루 마우스에서 혈관 누출을 평가 하는 프로토콜

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.

그대로 Glomeruli 성인 쥐 신장에서 분리 하는 효율적인 체질 하 방법

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