Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.
22 Related JoVE Articles!
Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment
Institutions: University Hospitals of Leicester.
The use of ex vivo
perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo
perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo
liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo
liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.
Medicine, Issue 82, Ex vivo, porcine, perfusion model, acid base balance, glucose, liver function, kidney function, cytokine response
Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels
Institutions: University of Duisburg-Essen Medical School.
The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The cerebral vasculature dynamically responds to various pathophysiological states and it exhibits considerable differences between strains and under conditions of genetic manipulations. Essentially, a reliable technique for intracranial vessel staining is essential in order to study the pathogenesis of ischemic stroke. Until recently, a set of different techniques has been employed to visualize the cerebral vasculature including injection of low viscosity resin, araldite F, gelatin mixed with various dyes1
carmine red, India ink) or latex with2
carbon black. Perfusion of white latex compound through the ascending aorta has been first reported by Coyle and Jokelainen3
. Maeda et al.2
have modified the protocol by adding carbon black ink to the latex compound for improved contrast visualization of the vessels after saline perfusion of the brain. However, inefficient perfusion and inadequate filling of the vessels are frequently experienced due to high viscosity of the latex compound4
. Therefore, we have described a simple and cost-effective technique using a mixture of two commercially available carbon black inks (CB1 and CB2) to visualize the cerebral vasculature in a reproducible manner5
. We have shown that perfusion with CB1+CB2 in mice results in staining of significantly smaller cerebral vessels at a higher density in comparison to latex perfusion5
. Here, we describe our protocol to identify the anastomotic points between the anterior (ACA) and middle cerebral arteries (MCA) to study vessel variations in mice with different genetic backgrounds. Finally, we demonstrate the feasibility of our technique in a transient focal cerebral ischemia model in mice by combining CB1+CB2-mediated vessel staining with TTC staining in various degrees of ischemic injuries.
Neuroscience, Issue 71, Neurobiology, Medicine, Anatomy, Physiology, Cellular Biology, Immunology, Neurology, Cerebral vascular anatomy, colored latex, carbon black, ink, stroke, vascular territories, brain, vessels, imaging, animal model
Use of a Hanging-weight System for Liver Ischemia in Mice
Institutions: University of Colorado, Denver, University of Colorado, Denver.
Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and can result in liver failure which has a high mortality rate1-2
. Therefore murine studies of hepatic ischemia have become an important field of research by providing the opportunity to utilize pharmacological and genetic studies3-9
. Specifically, conditional mice with tissue specific deletion of a gene (cre, flox system) provide insights into the role of proteins in particular tissues10-13
. Because of the technical difficulty associated with manually clamping the portal triad in mice, we performed a systematic evaluation using a hanging-weight system for portal triad occlusion which has been previously described3
. By using a hanging-weight system we place a suture around the left branch of the portal triad without causing any damage to the hepatic lobes, since also the finest clamps available can cause hepatic tissue damage because of the close location of liver tissue to the vessels. Furthermore, the right branch of the hepatic triad is still perfused thus no intestinal congestion occurs with this technique as blood flow to the right hepatic lobes is preserved. Furthermore, the portal triad is only manipulated once throughout the entire surgical procedure. As a result, procedures like pre-conditioning, with short times of ischemia and reperfusion, can be easily performed. Systematic evaluation of this model by performing different ischemia and reperfusion times revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine (ALT) and aspartate (AST) aminotransferase serum levels3,9
. Taken together, these studies confirm highly reproducible liver injury when using the hanging-weight system for hepatic ischemia and intermittent reperfusion. Thus, this technique might be useful for other investigators interested in liver ischemia studies in mice. Therefore the video clip provides a detailed step-by-step description of this technique.
Medicine, Issue 66, Physiology, Immunology, targeted gene deletion, murine model, liver failure, ischemia, reperfusion, video demonstration
Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET
Institutions: University of Münster, University of Münster, University of Münster.
The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function 1
. At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models.
Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR 2
. We herein present in addition small animal positron emission tomography (PET) using 18
F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation 3
. Further, this method can be applied for follow-up to improve monitoring of transplant rejection 4
Medicine, Issue 74, Molecular Biology, Biomedical Engineering, Bioengineering, Cellular Biology, Anatomy, Physiology, Immunology, Surgery, Tissue Engineering, Nephrology, transplantation, rat, kidney, renal, acute rejection, allograft, imaging, histology, positron emisson tomography, PET, 18F-fluorodeoxyglucose, FDG, rat, animal model
Orthotopic Hind-Limb Transplantation in Rats
Institutions: Innsbruck Medical University, University of Pittsburgh Medical Center.
Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.
JoVE Immunology, Issue 41, rat, hind limb, composite tissue, reconstructive transplantation
Establishment and Characterization of UTI and CAUTI in a Mouse Model
Institutions: Washington University School of Medicine.
Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli
(UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis
. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis
bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.
Medicine, Issue 100, Escherichia coli, UPEC, Enterococcus faecalis, uropathogenic, catheter, urinary tract infection, IBC, chronic cystitis
Mouse Kidney Transplantation: Models of Allograft Rejection
Institutions: The University of Edinburgh.
Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.
Medicine, Issue 92, transplantation, mouse model, surgery, kidney, immunology, rejection
Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles
Institutions: Heart Research Center Goettingen, University Medical Center Goettingen, German Center for Cardiovascular Research (DZHK) partner site Goettingen, University of Maryland School of Medicine.
In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+
release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.
Bioengineering, Issue 92, cardiac myocyte, atria, ventricle, heart, primary cell isolation, fluorescence microscopy, membrane tubule, transverse-axial tubule system, image analysis, image processing, T-tubule, collagenase
Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples
Institutions: University of Florence, University of Florence.
Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.
Medicine, Issue 86, cardiology, cardiac cells, electrophysiology, excitation-contraction coupling, action potential, calcium, myocardium, hypertrophic cardiomyopathy, cardiac patients, cardiac disease
An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart
Institutions: Eindhoven University of Technology, University Medical Center Utrecht.
Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro
and in vivo
experiments. The in vitro
experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo.
This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.
Bioengineering, Issue 100, supramolecular polymers, hydrogels, catheter injection, drug delivery, pH switchability, pig model
Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis
Institutions: University of California, San Francisco, University of California, San Francisco, University of California, San Francisco.
Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1
. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2
, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.
Neuroscience, Issue 57, vessel, vascular cast, capillary, cerebrovasculature, brain, blood, AVM, fistula
Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice
Institutions: Vanderbilt University Medical Center.
Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.
Medicine, Issue 78, Immunology, Infection, Biomedical Engineering, Anatomy, Physiology, Kidney, Mice, Inbred Strains, Renal Insufficiency, Acute Kidney Injury, Ischemia-reperfusion, acute kidney injury, post injury fibrosis, mice, ischemia, reperfusion, fibrosis, animal model
Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle
Institutions: University of Freiburg Medical Centre.
Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1
Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3
Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5
with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7
Gold standard for the in vivo
observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12
only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13
as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.
Medicine, Issue 66, Immunology, Physiology, Molecular Biology, microcirculation, ischemia-reperfusion injury, rat, cremaster muscle, leukocyte activation, intravital microscopy
Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90,
zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt
Institutions: Toronto General Hospital.
The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity.
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology.
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.
Medicine, Issue 99, Orthotopic Liver Transplantation, Hepatic, Porcine Model, Pig, Experimental, Transplantation, Graft Preservation, Ischemia Reperfusion Injury, Transplant Immunology, Bile Duct Reconstruction, Animal Handling
A Rat Model of Ventricular Fibrillation and Resuscitation by Conventional Closed-chest Technique
Institutions: Rosalind Franklin University of Medicine and Science.
A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic components of cardiopulmonary resuscitation in humans is herein described. The model was developed in 1988 and has been used in approximately 70 peer-reviewed publications examining a myriad of resuscitation aspects including its physiology and pathophysiology, determinants of resuscitability, pharmacologic interventions, and even the effects of cell therapies. The model featured in this presentation includes: (1) vascular catheterization to measure aortic and right atrial pressures, to measure cardiac output by thermodilution, and to electrically induce ventricular fibrillation; and (2) tracheal intubation for positive pressure ventilation with oxygen enriched gas and assessment of the end-tidal CO2
. A typical sequence of intervention entails: (1) electrical induction of ventricular fibrillation, (2) chest compression using a mechanical piston device concomitantly with positive pressure ventilation delivering oxygen-enriched gas, (3) electrical shocks to terminate ventricular fibrillation and reestablish cardiac activity, (4) assessment of post-resuscitation hemodynamic and metabolic function, and (5) assessment of survival and recovery of organ function. A robust inventory of measurements is available that includes – but is not limited to – hemodynamic, metabolic, and tissue measurements. The model has been highly effective in developing new resuscitation concepts and examining novel therapeutic interventions before their testing in larger and translationally more relevant animal models of cardiac arrest and resuscitation.
Medicine, Issue 98, Cardiopulmonary resuscitation, Hemodynamics, Myocardial ischemia, Rats, Reperfusion, Ventilation, Ventricular fibrillation, Ventricular function, Translational medical research
Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging
Institutions: Jena University Hospital, Friedrich-Schiller-University Jena, Jena University Hospital.
Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo
imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo
, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo
imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo
imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo
. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.
Bioengineering, Issue 95, Drug-delivery, Liposomes, Fluorochromes, Fluorescence-quenching, Optical imaging, Inflammation
Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice
Institutions: University of Texas Southwestern Medical Center, Washington University School of Medicine.
Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2
). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2
room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.
Medicine, Issue 99, Hypoxia, preconditioning, transient middle cerebral artery occlusion, stroke, neuroprotection, blood-brain barrier disruption
Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation
Institutions: University of Rochester, University of Rochester, University of Rochester Medical Center.
One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young’s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.
primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;
H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.
Chemistry, Issue 80, Poly(ethylene glycol), peptides, polymerization, polymers, methacrylation, peptide functionalization, 1H-NMR, MALDI-ToF, hydrogels, macromer synthesis
The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport
Institutions: Monash University (Parkville Campus).
The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g.,
diets, antigens, drugs) and in disease (e.g.,
inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.
Immunology, Issue 97, Intestine, Mesenteric, Lymphatic, Lymph, Carotid artery, Cannulation, Cannula, Rat, Drug, Lipid, Absorption, Surgery
Use of a Hanging-weight System for Isolated Renal Artery Occlusion
Institutions: University of Colorado, University of Colorado, Korea University College of Medicine.
In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3
. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1
. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4
. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5
. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6
. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6
.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1
. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13
. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14
. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.
Medicine, Issue 53, targeted gene deletion, murine model, acute renal failure, ischemia, reperfusion, video demonstration
Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology
Institutions: University of Pittsburgh School of Medicine, University of Pittsburgh School of Medicine.
We describe an in vitro
method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo
experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e.
smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo
. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro
smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.
Medicine, Issue 90, Krebs, species differences, in vitro, smooth muscle contractility, neural stimulation