Noi riconosciamo con gratitudine il supporto di una borsa di studio pilota del Fondo Stanford Research Pediatrica e K08DK087895-01 dal NIDDK.

1. Topo iniezione parete della vescica Scelta del ceppo di topi, l&#39;età e il sesso è dettata da esigenze sperimentali. Noi usiamo topi tra 8 e 12 settimane di età, dal momento che questo è una finestra di maturità immunologica prima senescenza. Come regola generale, i topi dovrebbero arrivare almeno una settimana prima di manipolazione sperimentale al fine di evitare fattori di confondimento indotta da stress. <ol><li> Pulire la superficie del tavolo operatorio con acqua e sapone. </li><li> Pulire la superficie del tavolo operatorio con Swipes Cide o salviette antisettico. </li><li> Autoclave pulizia degli strumenti chirurgici prima di utilizzare in chirurgia. </li><li> Inoltre, sterilizzare strumenti chirurgici con sterilizzatore a caldo tallone immediatamente prima dell&#39;uso, così come tra gli animali durante l&#39;intervento. </li><li> Pulire 100 microlitri siringhe Hamilton e 29 o 30 gauge (1 / 2 pollice di lunghezza), aghi da aspirazione ripetute e iniezione con alcool assoluto prima del primo utilizzo e, alla fine della procedura chirurgica definitiva. </li><li> Lavaggio e risciacquo siringhe Hamilton con sterilizzati PBS tra ogni animale. </li><li> Anestetizzare topi mettendoli in una camera di induzione isoflurano, con il set isoflurano tra 2-5%. </li><li> Una volta che l&#39;anestesia generale si ottiene, rimuovere il mouse dalla camera e posto in posizione supina con un batuffolo di caldo sotto per aiutare a mantenere la normale temperatura corporea. </li><li> Ottenere l&#39;anestesia manutenzione mettendo muso dell&#39;animale in un ugello che contiene vaporizzare isoflurano (titolato dal 1-3% se necessario per mantenere l&#39;anestesia del caso). </li><li> Rasatura della pelle addominale con Clippers. </li><li> Utilizzare un monouso, telino sterile chirurgico per coprire l&#39;ano per evitare la contaminazione fecale durante l&#39;intervento. </li><li> Utilizzare un secondo usa e getta, telino sterile chirurgico per coprire il campo operatorio (basso addome). </li><li> Prep all&#39;addome con tre pezzi di garza imbevuta di Betadine. Ripetere questa operazione tre volte. </li><li> Utilizzando un microscopio da dissezione di ingrandimento, fare abbassare un&#39;incisione addominale mediana con le forbici. </li><li> Esporre la vescica. </li><li> Se la vescica è piena, parzialmente da decomprimere delicata pressione verso il basso la cupola. </li><li> Iniettare la soluzione del campione (fino a 50 mL), con un calibro 29 o 30 aghi e siringhe, nella parete della cupola vescicale (iniezione intramurale) con la smussatura dell&#39;ago verso l&#39;alto. </li><li> Spingere lo stantuffo della siringa per iniettare la soluzione di esempio nella parete della vescica. A ben localizzato bleb è un&#39;indicazione di iniezione di successo delle cellule nella parete della vescica. </li><li> Rimuovere la siringa, chiudere l&#39;incisione con clip ferita e permettono di recuperare il mouse su un blocco del riscaldamento. </li></ol> 2. Rappresentante dei risultati: A ben localizzato bleb che non fluido perdite e resta stabile in dimensioni è un&#39;indicazione di iniezione di successo delle cellule nella parete della vescica (Figura 1). L&#39;analisi istologica può essere eseguito per confermare la presenza delle cellule iniettate nella parete della vescica. <img src="/files/ftp_upload/2523/2523fig1.jpg" alt="Figura 1"/> Figura 1. Esempio di iniezione di successo parete vescicale utilizzando inchiostro di china per l&#39;illustrazione. <img src="/files/ftp_upload/2523/2523fig2.jpg" alt="Figura 2"/> Figura 2. Schematica delle procedure sperimentali.

<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>

Nessun conflitto di interessi dichiarati.

Vescica iniezione del mouse parete permette l&#39;impianto di cellule specifiche in determinati settori della parete vescicale. Questa tecnica ha applicazioni gamma di modelli murini di cancro alla vescica, muscolo liscio, e biologia delle cellule staminali. Per definizione, la vescica di iniezione del mouse parete facilita l&#39;introduzione di cancro della vescica o di cellule staminali in una posizione ortotopico in modo che la loro crescita e differenziazione possono essere studiati in un contesto fisiologicamente r...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Isoflurano (Aerrane) </td><td> Baxter </td><td> NDC 10019-773-60 </td></tr><tr><td> 29 o 30 gauge aghi </td><td> Hamilton </td><td> 7803-06 e 7803-07 </td></tr><tr><td> 100 microlitri siringa </td><td> Hamilton </td><td> 7656-01 </td></tr></tbody></table>

mouse bladder wall injection

Vescica iniezione del mouse muro è una tecnica utile per lo studio dei fenomeni ortotopicamente vescica, tra cui cellule staminali, muscolo liscio, e la biologia del cancro. Prima di iniziare le iniezioni, l&#39;area chirurgica devono essere pulite con acqua e sapone e soluzione antisettica. Strumenti chirurgici devono essere sterilizzati prima dell&#39;uso e tra ogni animale. Ogni mouse è posta sotto anestesia per via inalatoria isoflurano (2-5% per l&#39;induzione, 1-3% per la manutenzione) e la sua vescica esposti facendo una incisione addominale mediana con le forbici. Se la vescica è piena, è parzialmente decompresso dalla spremitura delicata tra due dita. La sospensione di cellule di interesse è intramurale iniettato nella parete della cupola della vescica con un 29 o 30 gauge e 1 cc o più piccoli siringa. La ferita viene poi chiusa con clip ferita e il topo ha permesso di recuperare su un blocco del riscaldamento. Parete vescicale iniezione è una tecnica microchirurgica delicato che può essere masterizzato con la pratica.

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

Mouse Bladder Wall Injection

Vescica iniezione del mouse muro è un approccio utile per lo studio delle cellule staminali ortotopicamente della vescica e della biologia del cancro. Questo metodo microchirurgico delicato può essere padroneggiato con tecnica attenta e pratica.

Topo iniezione parete della vescica

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. Vescica iniezione del mouse muro è un approccio utile per lo studio delle cellule staminali ortotopicamente della vescica e della biologia del cancro. Questo metodo microchirurgico delicato può essere padroneggiato con tecnica attenta e pratica.

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.

Un modello ortotopico di cancro alla vescica Murine

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

Ricerca

Medicina

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Iperinsulinemico-euglicemico Morsetti in Cosciente, Mouse sfrenato

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

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.

L&#39;uso di cistometria in piccoli roditori: A Study of Chemosensation vescica

	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 ...

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.

Un modello ortotopico tumore della vescica e la valutazione del trattamento intravescicale sarna

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.

Iniezione sottoretinico di vettori di terapia genica e cellule staminali nel Eye mouse

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.

Riduzione del ventricolo sinistro stress di parete e di miglioramento della funzione in mancanza di Cuori con 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.

Un ortotopico cancro della vescica Modello di Gene Studi di consegna

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.

Mini-Invasiva Istituzione di murini ortotopico vescica Xenotrapianti

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.

Vescica urinaria Distensione Evocati visceromotoria Risposte come un modello per il dolore della vescica nei topi

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.

Tomografia ottica di coerenza di risoluzione ultraelevata Mouse per aiutare l'iniezione intraoculare nella ricerca retinica terapia genica

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