نحن الامتنان على الدعم من منحة رائدة من ستانفورد للأبحاث صندوق الأطفال و01 - K08DK087895 من جوائز كبيرة.

1. حقن المثانة الماوس الجدار وأملت اختيار سلالة الفأر ، والعمر ، والجنس ، وفقا لاحتياجات التجريبية. نستخدم الفئران بين 8 و 12 أسبوعا من العمر ، حيث أن هذه هي نافذة من النضج المناعية قبل الشيخوخة. كمبدأ عام ، يجب أن تصل الفئران على الأقل قبل أسبوع واحد للتلاعب التجريبية بغية تجنب التوتر الناجم عن عوامل خارجية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تنظيف سطح الطاولة الجراحية بالصابون والماء. </li><li style=";text-align:right;direction:rtl"> مسح سطح الطاولة الجراحية مع احدث الضربات الشديدة أو مناديل مطهرة. </li><li style=";text-align:right;direction:rtl"> الأوتوكلاف الأدوات الجراحية النظيفة قبل استخدامها في الجراحة. </li><li style=";text-align:right;direction:rtl"> بالإضافة إلى ذلك ، مع تعقيم الأدوات الجراحية معقمة حبة الساخنة مباشرة قبل استخدامها ، وكذلك بين الحيوانات أثناء الجراحة. </li><li style=";text-align:right;direction:rtl"> محاقن نظيفة 100 ميكرولتر هاميلتون و 29 أو 30 مقياس (1 / 2 بوصة طويلة) بواسطة الإبر والحقن التطلع المتكررة مع الكحول المطلق قبل الاستخدام لأول مرة وعند نهاية الإجراء الجراحي النهائي. </li><li style=";text-align:right;direction:rtl"> غسل وشطف المحاقن المعقمة هاميلتون مع الفوسفات مخزنة المالحة بين كل الحيوانات. </li><li style=";text-align:right;direction:rtl"> تخدير الفئران عن طريق وضعها في غرفة isoflurane الاستقراء ، مع مجموعة isoflurane بين 2-5 ٪. </li><li style=";text-align:right;direction:rtl"> متى تم تحقيق التخدير العام ، وإزالة الماوس من الغرفة ووضعه في موقف ضعيف مع وسادة دافئة تحت للمساعدة في الحفاظ درجة حرارة الجسم طبيعية. </li><li style=";text-align:right;direction:rtl"> تحقيق التخدير الصيانة عن طريق وضع أنف الحيوان في فوهة تحتوي على تبخير isoflurane (معاير من 1-3 ٪ حسب الضرورة للحفاظ على التخدير المناسبة). </li><li style=";text-align:right;direction:rtl"> حلق جلد البطن مع لوس انجليس كليبرز. </li><li style=";text-align:right;direction:rtl"> استخدام ، ثنى الجراحية المعقمة المتاح لتغطية فتحة الشرج لمنع تلوث برازي أثناء الجراحة. </li><li style=";text-align:right;direction:rtl"> استخدام الثاني ، ثنى الجراحية المعقمة المتاح لتغطية الجراحي الميداني (أسفل البطن). </li><li style=";text-align:right;direction:rtl"> الإعدادية في البطن مع ثلاث قطع من الشاش Betadine غارقة. كرر هذه الخطوة ثلاث مرات. </li><li style=";text-align:right;direction:rtl"> باستخدام مجهر تشريح للتضخم ، وجعل الدنيا شق البطن خط الوسط مع المقص. </li><li style=";text-align:right;direction:rtl"> تعرض المثانة. </li><li style=";text-align:right;direction:rtl"> إذا كانت المثانة ممتلئة ، ضغط جزئيا من قبل الضغط النزولي لطيف في القبة. </li><li style=";text-align:right;direction:rtl"> حقن محلول العينة (تصل إلى 50 ميكرولتر) ، وذلك باستخدام إبرة قياس 29 أو 30 والمحاقن ، في جدار القبة المثانة (حقن جماعية) مع شطبة من الإبرة التي تواجه صعودا. </li><li style=";text-align:right;direction:rtl"> دفع المكبس من حقنة لحقن محلول العينة في جدار المثانة. وفقاعة جيدا المترجمة هو مؤشر على نجاح حقن الخلايا في جدار المثانة. </li><li style=";text-align:right;direction:rtl"> إزالة المحاقن ، وإغلاق شق الجرح مع لقطات والسماح الماوس لاستعادة العافية الاقتصادية على منصة ارتفاع حرارة الارض. </li></ol> 2. ممثل النتائج : وفقاعة جيدا المترجمة التي لا تسرب السوائل ويبقى مستقرا في الحجم هو دليل على نجاح حقن الخلايا في جدار المثانة (الشكل 1). ويمكن إجراء التحليل النسيجي للتأكد من وجود حقن الخلايا في جدار المثانة. <img src="/files/ftp_upload/2523/2523fig1.jpg" alt="الشكل 1"/> الشكل 1. مثال لنجاح حقن المثانة الحائط باستخدام الحبر الهند التوضيح. <img src="/files/ftp_upload/2523/2523fig2.jpg" alt="الشكل 2"/> الشكل 2. تخطيطي الإجراءات التجريبية.

<ol>
	<li>Dinney, C. P., Fishbeck, R., Singh, R. K., Eve, B., Pathak, S., Brown, N., Xie, B., Fan, D., Bucana, C., Fidler, I. J., Killion, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Metastatic+Variants+from+Human+Transitional+Cell+Carcinoma+Passaged+by+Orthotopic+Implantation+in+Athymic+Nude+Mice.">Isolation and Characterization of Metastatic Variants from Human Transitional Cell Carcinoma Passaged by Orthotopic Implantation in Athymic Nude Mice.</a> The Journal of Urology. 154, 1532-1538 (1995).</li><li>Singh, A. V., Franke, A. A., Blackburn, G. L., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soy+Phytochemicals+Prevent+Orthotopic+Growth+and+Metastasis+of+Bladder+Cancer+in+Mice+by+Alterations+of+Cancer+Cell+Proliferation+and+Apoptosis+and+Tumor+Angiogenesis.">Soy Phytochemicals Prevent Orthotopic Growth and Metastasis of Bladder Cancer in Mice by Alterations of Cancer Cell Proliferation and Apoptosis and Tumor Angiogenesis.</a> Cancer Res. 66, 1851-1858 (2006).</li><li>Yanagiuchi, A., Miyake, H., Nomi, M., Takenaka, A., Fujisawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+Microenvironment+by+Growth+Factors+Regulates+the+In+Vivo+Growth+of+Skeletal+Myoblasts.">Modulation of the Microenvironment by Growth Factors Regulates the In Vivo Growth of Skeletal Myoblasts.</a> BJU International. 103, 1569-1573 (2009).</li><li>Miyake, H., Hara, I., Yamanaka, K., Gohji, K., Arakawa, S., Kamidono, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Bcl-2+Enhances+Metastatic+Potential+of+Human+Bladder+Cancer+Cells.">Overexpression of Bcl-2 Enhances Metastatic Potential of Human Bladder Cancer Cells.</a> British Journal of Cancer. 79, 1651-1656 (1999).</li><li>Chancellor, M. B., Yokoyama, T., Tirney, S., Mattes, C. E., Ozawa, H., Yoshimura, N., Groat, W. C. d. e., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+Results+of+Myoblast+Injection+into+the+Urethra+and+Bladder+Wall:+a+Possible+method+for+the+Treatment+of+Stress+Urinary+Incontinence+and+Impaired+Detrusor+Contractility.">Preliminary Results of Myoblast Injection into the Urethra and Bladder Wall: a Possible method for the Treatment of Stress Urinary Incontinence and Impaired Detrusor Contractility.</a> Neurourol Urodyn. 19, 279-287 (2000).</li><li>Dinney, C. P., Tanguay, S., Bucana, C. D., Eve, B. Y., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravesical+Liposomal+Muramyl+Tripeptide+Phosphatidylethanolamine+Treatment+of+Human+Bladder+Carcinoma+Growing+in+Nude+Mice.">Intravesical Liposomal Muramyl Tripeptide Phosphatidylethanolamine Treatment of Human Bladder Carcinoma Growing in Nude Mice.</a> J Interferon Cytokine Res. 15, 585-592 (1995).</li><li>Mohamedali, K. A., Kedary, D., Sweeney, P., Kamaty, A., Davisy, D. W., Evey, B. Y., Huangy, S., Thorpez, P. E., Dinney, C. P., Rosenblum, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Vascular-targeting+Fusion+toxin+VEGF121/rGel+Inhibits+the+Growth+of+Orthotopic+Human+Bladder+Carcinoma+Tumors.">The Vascular-targeting Fusion toxin VEGF121/rGel Inhibits the Growth of Orthotopic Human Bladder Carcinoma Tumors.</a> Neoplasia. 7, 912-920 (2005).</li><li>Slaton, J. W., Perrotte, P., Inoue, K., Dinney, C. P., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-&#945;-mediated+Down-Regulation+of+Angiogenesis-related+Genes+and+Therapy+of+Bladder+Cancer+Are+Dependent+on+Optimization+of+Biological+Dose+and+Schedule.">Interferon-&#945;-mediated Down-Regulation of Angiogenesis-related Genes and Therapy of Bladder Cancer Are Dependent on Optimization of Biological Dose and Schedule.</a> Clinical Cancer Research. 5, 2726-2734 (1999).</li><li>Yokoyama, T., Huard, J., Pruchnic, R., Yoshimura, N., Qu, Z., Cao, B., De Groat, W. C., Kumon, H., Chancellor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle-Derived+Cell+Transplantation+and+Differentiation+into+Lower+Urinary+Tract+Smooth+Muscle.">Muscle-Derived Cell Transplantation and Differentiation into Lower Urinary Tract Smooth Muscle.</a> Urology. 57, 826-831 (2001).</li><li>Chan, E., Patel, A., Heston, W., Larchian, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Orthotopic+Models+for+Bladder+Cancer+Research.">Mouse Orthotopic Models for Bladder Cancer Research.</a> BJU International. 104, 1286-1291 (1988).</li><li>Russell, P. J., Jelbart, M., Wills, E., Singh, S., Wass, J., Witherspoon, J., Raghavan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+new+human+bladder+cancer+cell+line+showing+features+of+squamous+and+glandular+differentiation.">Establishment and characterization of a new human bladder cancer cell line showing features of squamous and glandular differentiation.</a> Int. J. Cancer. 41, 74-74 (1988).</li><li>Ahlering, T. E., Dubeau, L., Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+in+vivo+model+to+study+invasion+and+metastasis+of+human+bladder+carcinoma.">A new in vivo model to study invasion and metastasis of human bladder carcinoma.</a> Cancer Res. 41, 6660-6660 (1987).</li><li>Heaney, J. A., Omellas, E. P., Daly, J. J., Lin, J. C., Rout, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+growth+of+human+bladder+cancer+cell+lines.">In vivo growth of human bladder cancer cell lines.</a> Invest. Urol. 15, 380-380 (1978).</li><li>Hadaschik, B., Black, P., Sea, J., Metwalli, A., Fazli, L., Dinney, C., Gleave, M., So, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+validated+mouse+model+for+orthotopic+bladder+cancer+using+transurethral+tumor+inoculation+and+bioluminescence+imaging.">A validated mouse model for orthotopic bladder cancer using transurethral tumor inoculation and bioluminescence imaging.</a> BJU International. 100, 1377-1384 (2007).</li></ol>

الإعلان عن أي تضارب في المصالح.

حقن الفأر جدار المثانة يسمح للزرع خلايا معينة في مناطق محددة من جدار المثانة. هذه التقنية لها تطبيقات واسعة لنماذج الماوس من مرض سرطان المثانة ، العضلات الملساء ، وبيولوجيا الخلايا الجذعية. بحكم التعريف ، وحقن الماوس جدار المثانة يسهل إدخال سرطان المثانة أو الخلايا ...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td></tr><tr><td> Isoflurane (Aerrane) </td><td> باكستر </td><td> NDC 10019-773-60 </td></tr><tr><td> الإبر قياس 29 أو 30 </td><td> هاميلتون </td><td> 7803-06 أو 7803-07 </td></tr><tr><td> 100 ميكروليتر حقنة </td><td> هاميلتون </td><td> 7656-01 </td></tr></tbody></table>

mouse bladder wall injection

حقن الفأر جدار المثانة هي تقنية مفيدة لدراسة الظواهر orthotopically المثانة ، بما في ذلك الخلايا الجذعية ، العضلات الملساء ، وعلم الأحياء السرطان. قبل البدء في الحقن ، يجب تنظيف المنطقة الجراحية بالماء والصابون ومحلول مطهر. يجب تعقيم المعدات الجراحية قبل الاستخدام ، وبين كل حيوان. يتم وضع كل الماوس تحت التخدير isoflurane استنشاقه (2-5 ٪ للتحريض ، 1-3 ٪ للصيانة) والمثانة لها كشفها بواسطة صنع شق خط الوسط في البطن مع المقص. إذا كانت المثانة ممتلئة ، فمن ضغط جزئيا لطيف الضغط بين إصبعين. يتم حقن intramurally تعليق خلية من الاهتمام في جدار القبة المثانة باستخدام إبرة قياس 29 أو 30 و 1 سم أو أصغر المحاقن. ثم يتم إغلاق الجرح باستخدام مقاطع الجرح وسمح لاسترداد الماوس على لوحة الاحتباس الحراري. حقن المثانة الجدار هو أسلوب المجهرية الدقيقة التي يمكن أن يتقن مع الممارسة.

Watch this Scientific Journal Video about Mouse Bladder Wall Injection at JoVE.com

Mouse Bladder Wall Injection

حقن الفأر جدار المثانة هو نهج مفيد لدراسة orthotopically المثانة الخلايا الجذعية وعلم الأحياء السرطان. يمكن أن يتقن هذه الطريقة المجهرية الدقيقة مع تقنية دقيقة والممارسة.

حقن المثانة الماوس الجدار

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

Research

JoVE Journal

Medicine

23.8K Views.  Stanford University School of Medicine. حقن الفأر جدار المثانة هو نهج مفيد لدراسة orthotopically المثانة الخلايا الجذعية وعلم الأحياء السرطان. يمكن أن يتقن هذه الطريقة المجهرية الدقيقة مع تقنية دقيقة والممارسة.

Video: Mouse Bladder Wall Injection

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

نموذج مثلي من سرطان المثانة الفئران

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

استعمال قياس المثانة في القوارض الصغيرة: دراسة في Chemosensation المثانة

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-&kappa;B Reporter Transgenic

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as pre-clinical models to test interventions. In these methods, we describe a technique in which lipopolysaccharide (LPS) is injected into the lactating mouse mammary gland via the nipple, effectively modeling mastitis, or inflammation, of the gland. This simulated infection results in increased nuclear factor kappa B (NF-&kappa;B) signaling, as visualized through bioluminescent imaging of an NF-&kappa;B luciferase reporter mouse1.
Our ultimate goal in developing these methods was to study the inflammation associated with mastitis in the lactating gland, which often includes redness, swelling, and immune cell infiltration2,3. Therefore, we were keenly aware that incision or any type of wounding of the skin, the nipple, or the gland in order to introduce the LPS could not be utilized in our methods since the approach would likely confound the read-out of inflammation. We also desired a straight-forward method that did not require specially made hand-drawn pipettes or the use of micromanipulators to hold these specialized tools in place. Thus, we determined to use a commercially available insulin syringe and to inject the agent into the mammary duct of an intact nipple. This method was successful and allowed us to study the inflammation associated with LPS injection without any additional effects overlaid by the process of injection. In addition, this method also utilized an NF-&kappa;B luciferase reporter transgenic mouse and bioluminescent imaging technology to visually and quantitatively show increased NF-&kappa;B signaling within the LPS-injected gland4.
These methods are of interest to researchers of many disciplines who wish to model disease within the lactating mammary gland, as ultimately, the technique described here could be utilized for injection of a number of substances, and is not limited to only LPS.

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-&amp;kappa;B Reporter Transgenic

Described here is a technique in which lipopolysaccharide is injected into the lactating mouse mammary gland via the nipple to simulate mastitis, a condition commonly caused by bacterial infection. Lipopolysaccharide injection results in increased nuclear factor kappa B (NF-&kappa;B) signaling, visualized through bioluminescent imaging of an NF-&kappa;B luciferase reporter mouse.

تصور اشارة عبر مراسل المعدلة وراثيا NF-كيلوبايت: حقن ينترادوكتال من LPS ونموذج الفأر من التهاب الضرع

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

ورم في المثانة Orthotopic النموذجي وتقييم المعالجة سارنا داخل المثانة

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

تحت الشبكية حقن المتجهات العلاج الجيني والخلايا الجذعية في العين ماوس

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

تخفيض في البطين الأيسر الإجهاد الحائط والتحسين في وظيفة في عدم قلوب باستخدام Algisyl-LVR

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

وسرطان المثانة نموذج مثلي للدراسات توصيل الجينات

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

مؤسسة المناظير الفئران مثلي المثانة Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

الردود المثانة البولية انتفاخ مستدعى الحشوي الحركي نموذجا لألم المثانة في الفئران

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

القرار أولتراهيغ الماوس الضوئية التماسك التصوير المقطعي لمساعدة الحقن داخل مقلة العين في أبحاث العلاج الجيني الشبكية

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...