A Decentralized (Ex Vivo) Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

10.3791/60344 ◽  
2019 ◽  
Author(s):  
Leonie Durnin ◽  
Robert D. Corrigan ◽  
Kenton M. Sanders ◽  
Violeta N. Mutafova-Yambolieva
Keyword(s):  
Ex Vivo ◽  
Direct Access ◽  
Detrusor Muscle ◽  
Bladder Filling ◽  
Bladder Model

scholarly journals Urothelial purine release during filling of murine and primate bladders

2016 ◽  
Vol 311 (4) ◽  
pp. F708-F716 ◽  
Author(s):  
Leonie Durnin ◽  
Sebastien Hayoz ◽  
Robert D. Corrigan ◽  
Andrew Yanez ◽  
Sang Don Koh ◽  
...  
Keyword(s):  
High Performance ◽  
Ex Vivo ◽  
Bladder Wall ◽  
Cell Types ◽  
Physiological Solution ◽  
Biologically Active ◽  
Detrusor Muscle ◽  
Bladder Filling ◽  
Bladder Urothelium ◽  
Bladder Sensation

During urinary bladder filling the bladder urothelium releases chemical mediators that in turn transmit information to the nervous and muscular systems to regulate sensory sensation and detrusor muscle activity. Defects in release of urothelial mediators may cause bladder dysfunctions that are characterized with aberrant bladder sensation during bladder filling. Previous studies have demonstrated release of ATP from the bladder urothelium during bladder filling, and ATP remains the most studied purine mediator that is released from the urothelium. However, the micturition cycle is likely regulated by multiple purine mediators, since various purine receptors are found present in many cell types in the bladder wall, including urothelial cells, afferent nerves, interstitial cells in lamina propria, and detrusor smooth muscle cells. Information about the release of other biologically active purines during bladder filling is still lacking. Decentralized bladders from C57BL/6 mice and Cynomolgus monkeys ( Macaca fascicularis) were filled with physiological solution at different rates. Intraluminal fluid was analyzed by high-performance liquid chromatography with fluorescence detection for simultaneous evaluation of ATP, ADP, AMP, adenosine, nicotinamide adenine dinucleotide (NAD+), ADP-ribose, and cADP-ribose content. We also measured ex vivo bladder filling pressures and performed cystometry in conscious unrestrained mice at different filling rates. ATP, ADP, AMP, NAD+, ADPR, cADPR, and adenosine were detected released intravesically at different ratios during bladder filling. Purine release increased with increased volumes and rates of filling. Our results support the concept that multiple urothelium-derived purines likely contribute to the complex regulation of bladder sensation during bladder filling.

scholarly journals Convective forces increase rostral delivery of intrathecal antisense oligonucleotides in the cynomolgus monkey nervous system

2020 ◽  
Author(s):  
Jenna M. Sullivan ◽  
Curt Mazur ◽  
Daniel A. Wolf ◽  
Laura Horky ◽  
Nicolas Currier ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Ex Vivo ◽  
High Volume ◽  
Radioactive Tracer ◽  
Direct Access ◽  
Cynomolgus Monkeys ◽  
Added Benefit ◽  
The Subject ◽  
Dose Volumes ◽  
Tracer Molecules

ABSTRACTBackgroundThe intrathecal (IT) dosing route introduces drugs directly into the CSF to bypass the blood-brain barrier and gain direct access to the CNS. We evaluated the use of convective forces acting on the cerebrospinal fluid as a means for increasing rostral delivery of IT dosed radioactive tracer molecules and antisense oligonucleotides (ASO) in the monkey CNS. We also measured the cerebral spinal fluid (CSF) volume in a group of cynomolgus monkeys.MethodsThere are three studies presented, in each of which cynomolgus monkeys were injected into the IT space with radioactive tracer molecules and/or ASO by lumbar puncture in either a low or high volume. The first study used the radioactive tracer 64Cu-DOTA and PET imaging to evaluate the effect of the convective forces. The second study combined the injection of the radioactive tracer 99mTc-DTPA and ASO, then used SPECT imaging and ex vivo tissue analysis of the effects of convective forces to bridge between the tracer and the ASO distributions. The third experiment evaluated the effects of different injection volumes on the distribution of an ASO. In the course of performing these studies we also measured the CSF volume in the subject monkeys by Magnetic Resonance Imaging.ResultsIt was consistently found that larger bolus dose volumes produced greater rostral distribution along the neuraxis. Thoracic percussive treatment also increased rostral distribution of low volume injections. There was little added benefit on distribution by combining the thoracic percussive treatment with the high-volume injection. The CSF volume of the monkeys was found to be 11.9 ± 1.6 cm3.ConclusionsThese results indicate that increasing convective forces after IT injection increases distribution of molecules up the neuraxis. In particular, the use of high IT injection volumes will be useful to increase rostral CNS distribution of therapeutic ASOs for CNS diseases in the clinic.

scholarly journals The MEMIC: An ex vivo system to model the complexity of the tumor microenvironment

2021 ◽  
Author(s):  
Libuše Janská ◽  
Libi Anandi ◽  
Nell C. Kirchberger ◽  
Zoran S. Marinkovic ◽  
Logan T. Schachtner ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Cells ◽  
Ex Vivo ◽  
Tumor Stroma ◽  
Stem Cell Differentiation ◽  
Blood Stream ◽  
Direct Access ◽  
Ex Vivo Model

There is an urgent need for accurate, scalable, and cost-efficient experimental systems to model the complexity of the tumor microenvironment. Here, we detail how to fabricate and use the Metabolic Microenvironment Chamber (MEMIC) – a 3D-printed ex vivo model of intratumoral heterogeneity. A major driver of the cellular and molecular diversity in tumors is the accessibility to the blood stream that provides key resources such as oxygen and nutrients. While some tumor cells have direct access to these resources, many others must survive under progressively more ischemic environments as they reside further from the vasculature. The MEMIC is designed to simulate the differential access to nutrients and allows co-culturing different cell types, such as tumor and immune cells. This system is optimized for live imaging and other microscopy-based approaches, and it is a powerful tool to study tumor features such as the effect of nutrient scarcity on tumor-stroma interactions. Due to its adaptable design and full experimental control, the MEMIC provide insights into the tumor microenvironment that would be difficult to obtain via other methods. As a proof of principle, we show that cells sense gradual changes in metabolite concentration resulting in multicellular spatial patterns of signal activation and cell proliferation. To illustrate the ease of studying cell-cell interactions in the MEMIC, we show that ischemic macrophages reduce epithelial features in neighboring tumor cells. We propose the MEMIC as a complement to standard in vitro and in vivo experiments, diversifying the tools available to accurately model, perturb, and monitor the tumor microenvironment, as well as to understand how extracellular metabolites affect other processes such as wound healing and stem cell differentiation.

scholarly journals Multiple Consecutive Lavage Samplings Reveal Greater Burden of Disease and Provide Direct Access to the Nontypeable Haemophilus influenzae Biofilm in Experimental Otitis Media

2007 ◽  
Vol 75 (8) ◽  
pp. 4158-4172 ◽  
Author(s):  
Magali Leroy ◽  
Howard Cabral ◽  
Marisol Figueira ◽  
Valérie Bouchet ◽  
Heather Huot ◽  
...  
Keyword(s):  
Gene Expression ◽  
Haemophilus Influenzae ◽  
Otitis Media ◽  
Vaccine Development ◽  
Ex Vivo ◽  
Direct Analysis ◽  
Direct Access ◽  
Nontypeable Haemophilus Influenzae ◽  
Nasopharyngeal Colonization

ABSTRACT The typically recovered quantity of nontypeable Haemophilus influenzae (NTHi) bacteria in an ex vivo middle ear (ME) aspirate from the chinchilla model of experimental otitis media is insufficient for direct analysis of gene expression by microarray or of lipopolysaccharide glycoforms by mass spectrometry. This prompted us to investigate a strategy of multiple consecutive lavage samplings to increase ex vivo bacterial recovery. As multiple consecutive lavage samples significantly increased the total number of bacterial CFU collected during nasopharyngeal colonization or ME infection, this led us to evaluate whether bacteria sequentially acquired from consecutive lavages were similar. Comparative observation of complete ex vivo sample series by microscopy initially revealed ME inflammatory fluid consisting solely of planktonic-phase NTHi. In contrast, subsequent lavage samplings of the same infected ear revealed the existence of bacteria in two additional growth states, filamentous and biofilm encased. Gene expression analysis of such ex vivo samples was in accord with different bacterial growth phases in sequential lavage specimens. The existence of morphologically distinct NTHi subpopulations with varying levels of gene expression indicates that the pooling of specimens requires caution until methods for their separation are developed. This study based on multiple consecutive lavages is consistent with prior reports that NTHi forms a biofilm in vivo, describes the means to directly acquire ex vivo biofilm samples without sacrificing the animal, and has broad applicability for a study of mucosal infections. Moreover, this approach revealed that the actual burden of bacteria in experimental otitis media is significantly greater than was previously reported. Such findings may have direct implications for antibiotic treatment and vaccine development against NTHi.

scholarly journals Phases of decompensation during acute ischemia demonstrated in an ex vivo porcine bladder model

2020 ◽  
Vol 9 (5) ◽  
pp. 2138-2145
Author(s):  
Natalie R. Swavely ◽  
Zachary E. Cullingsworth ◽  
Naveen Nandanan ◽  
John E. Speich ◽  
Adam P. Klausner
Keyword(s):  
Ex Vivo ◽  
Acute Ischemia ◽  
Bladder Model

scholarly journals Nerve transfer for restoration of lower motor neuron-lesioned bladder function. Part 3: Correlation between histological changes and nerve evoked contractions

2021 ◽  
Author(s):  
Mary F Barbe ◽  
Courtney L Testa ◽  
Geneva E. Cruz ◽  
Nagat Frara ◽  
Ekta Tiwari ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Contractile Function ◽  
Ex Vivo ◽  
Bladder Function ◽  
Detrusor Muscle ◽  
Axon Density ◽  
Spinal Roots ◽  
One Year ◽  
Intramural Ganglia ◽  
Stimulation Of

We determined the effect of pelvic organ decentralization and reinnervation one year later on urinary bladder histology and function. Nineteen canines underwent decentralization by bilateral transection of all coccygeal and sacral (S) spinal roots, dorsal roots of lumbar (L)7 and hypogastric nerves. After exclusions, 8 were reinnervated 12 months post-decentralization with obturator-to-pelvic and sciatic-to-pudendal nerve transfers, then euthanized 8-12 months later; four served as long-term decentralized only animals. Before euthanasia, pelvic or transferred nerves and L1-S3 spinal roots were stimulated and maximum detrusor pressure (MDP) recorded. Bladder specimens were collected for histological and ex vivo smooth muscle contractility studies. Both reinnervated and decentralized animals showed less or denuded urothelium, fewer intramural ganglia, and more inflammation and collagen, than controls, although percent muscle was maintained. In reinnervated animals, pgp9.5+ axon density was higher, compared to decentralized animals. Ex vivo smooth muscle contractions in response to KCl correlated positively with submucosal inflammation, detrusor muscle thickness, pgp9.5+ axon density. In vivo, reinnervated animals showed higher MDP after stimulation of L1-L6 roots, compared to their transected L7-S3 roots, and reinnervated and decentralized animals showed lower MDP than controls after stimulation of nerves (due likely to fibrotic nerve encapsulation). MDP correlated negatively with detrusor collagen and inflammation, and positively with pgp9.5+ axon density and intramural ganglia numbers. These results demonstrate that bladder function can be improved by transfer of obturator nerves to pelvic nerves at one year after decentralization, although the fibrosis and inflammation that developed were associated with decreased contractile function.

scholarly journals Identification of Receptor Ligands by Screening Phage-Display Peptide LibrariesEx Vivoon Microdissected Kidney Tubules

2001 ◽  
Vol 12 (2) ◽  
pp. 308-316 ◽  
Author(s):  
ALEX ODERMATT ◽  
ANNETTE AUDIGÉ ◽  
CHRISTOPH FRICK ◽  
BRUNO VOGT ◽  
BRIGITTE M. FREY ◽  
...  
Keyword(s):  
Phage Display ◽  
Ex Vivo ◽  
Specific Binding ◽  
Cell System ◽  
Membrane Receptors ◽  
Direct Access ◽  
Kidney Tubules ◽  
Receptor Ligands ◽  
Renal Epithelial Cell ◽  
Linear Peptide

Abstract. A novel method to identify receptor ligands for defined renal tubular segments has been developed.Ex vivoscreening of phage-display peptide libraries on isolated intact rat proximal convoluted tubules (PCT) and cortical collecting ducts (CCD) allowed the direct access of phage to the basolateral surface of tubular epithelial cells. Two distinct peptide motifs were selected for CCD and PCT, indicating differential expression of some membrane receptors on the basolateral surface of defined kidney tubule segments. Using the linear peptide motif ELRGD(R/M)AX(W/L), recovered from freshly isolated rat CCD, mediated 16-fold selectivity of phage binding to CCD compared with PCT. Binding to CCD was 39-fold higher than that of a random control phage. Binding and subsequent internalization of phage, most likely by an integrin-mediated endocytosis pathway, was abolished by the addition of the corresponding synthetic peptide. Furthermore, the results demonstrate that presentation and flanking amino acids determine the specific binding properties of RGD ligands to their putative integrin receptors. The results emphasize the need of a native cell system for the identification of renal epithelial cell surface ligands. Such ligands are of potential relevance for the analysis of interactions between extracellular matrix and kidney tubules or for the development of improved vectors for kidney-specific drug delivery or gene transfer.

Potential vascular mechanisms in an ex vivo functional pig bladder model

2018 ◽  
Vol 37 (8) ◽  
pp. 2425-2433 ◽  
Author(s):  
Uzoma A. Anele ◽  
Paul H. Ratz ◽  
Andrew F. Colhoun ◽  
Sydney Roberts ◽  
Ryan Musselman ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Bladder Model

Ex vivo deformations of the urinary bladder wall during whole bladder filling: Contributions of extracellular matrix and smooth muscle

2010 ◽  
Vol 43 (9) ◽  
pp. 1708-1716 ◽  
Author(s):  
Aron Parekh ◽  
Alexander D. Cigan ◽  
Silvia Wognum ◽  
Rebecca L. Heise ◽  
Michael B. Chancellor ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Urinary Bladder ◽  
Ex Vivo ◽  
Bladder Wall ◽  
Bladder Filling ◽  
Urinary Bladder Wall

scholarly journals Development of P301S tau seeded organotypic hippocampal slice cultures to study potential therapeutics

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
James M. McCarthy ◽  
Jasmeet Virdee ◽  
Jessica Brown ◽  
Daniel Ursu ◽  
Zeshan Ahmed ◽  
...  
Keyword(s):  
Hippocampal Slice ◽  
Ex Vivo ◽  
Direct Access ◽  
In Vivo Studies ◽  
Dependent Manner ◽  
Ex Vivo Model ◽  
Concentration Dependent Manner ◽  
Hippocampal Slice Cultures ◽  
Organotypic Hippocampal Slice Cultures

AbstractIntracellular tau inclusions are a pathological hallmark of Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration and other sporadic neurodegenerative tauopathies. Recent in vitro and in vivo studies have shown that tau aggregates may spread to neighbouring cells and functionally connected brain regions, where they can seed further tau aggregation. This process is referred to as tau propagation. Here we describe an ex vivo system using organotypic hippocampal slice cultures (OHCs) which recapitulates aspects of this phenomenon. OHCs are explants of hippocampal tissue which may be maintained in culture for months. They maintain their synaptic connections and multicellular 3D architecture whilst also permitting direct control of the environment and direct access for various analysis types. We inoculated OHCs prepared from P301S mouse pups with brain homogenate from terminally ill P301S mice and then examined the slices for viability and the production and localization of insoluble phosphorylated tau. We show that following seeding, phosphorylated insoluble tau accumulate in a time and concentration dependent manner within OHCs. Furthermore, we show the ability of the conformation dependent anti-tau antibody, MC1, to compromise tau accrual in OHCs, thus showcasing the potential of this therapeutic approach and the utility of OHCs as an ex vivo model system for assessing such therapeutics.

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