Experimental saccular aneurysm models are necessary for testing novel surgical and endovascular treatment options and devices before they are introduced into clinical practice. Furthermore, experimental models are needed to elucidate the complex aneurysm biology leading to rupture of saccular aneurysms.
Several different kinds of experimental models for saccular aneurysms have been established in different species. Many of them, however, require special skills, expensive equipment, or special environments, which limits their widespread use. A simple, robust, and inexpensive experimental model is needed as a standardized tool that can be used in a standardized manner in various institutions.
The microsurgical rat abdominal aortic sidewall aneurysm model combines the possibility to study both novel endovascular treatment strategies and the molecular basis of aneurysm biology in a standardized and inexpensive manner. Standardized grafts by means of shape, size, and geometry are harvested from a donor rat's descending thoracic aorta and then transplanted to a syngenic recipient rat. The aneurysms are sutured end-to-side with continuous or interrupted 9-0 nylon sutures to the infrarenal abdominal aorta.
We present step-by-step procedural instructions, information on necessary equipment, and discuss important anatomical and surgical details for successful microsurgical creation of an abdominal aortic sidewall aneurysm in the rat.
23 Related JoVE Articles!
Small Bowel Transplantation In Mice
Institutions: University of California, San Francisco - UCSF.
Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.
Issue 7, Immunology, Transplantation, Transplant Rejection, Small Bowel
A Novel Rescue Technique for Difficult Intubation and Difficult Ventilation
Institutions: Children’s Hospital of Michigan, St. Jude Children’s Research Hospital.
We describe a novel non surgical technique to maintain oxygenation and ventilation in a case of difficult intubation and difficult ventilation, which works especially well with poor mask fit.
Can not intubate, can not ventilate" (CICV) is a potentially life threatening situation. In this video we present a simulation of the technique we used in a case of CICV where oxygenation and ventilation were maintained by inserting an endotracheal tube (ETT) nasally down to the level of the naso-pharynx while sealing the mouth and nares for successful positive pressure ventilation.
A 13 year old patient was taken to the operating room for incision and drainage of a neck abcess and direct laryngobronchoscopy. After preoxygenation, anesthesia was induced intravenously. Mask ventilation was found to be extremely difficult because of the swelling of the soft tissue. The face mask could not fit properly on the face due to significant facial swelling as well. A direct laryngoscopy was attempted with no visualization of the larynx. Oxygen saturation was difficult to maintain, with saturations falling to 80%. In order to oxygenate and ventilate the patient, an endotracheal tube was then inserted nasally after nasal spray with nasal decongestant and lubricant. The tube was pushed gently and blindly into the hypopharynx. The mouth and nose of the patient were sealed by hand and positive pressure ventilation was possible with 100% O2
with good oxygen saturation during that period of time. Once the patient was stable and well sedated, a rigid bronchoscope was introduced by the otolaryngologist showing extensive subglottic and epiglottic edema, and a mass effect from the abscess, contributing to the airway compromise. The airway was secured with an ETT tube by the otolaryngologist.This video will show a simulation of the technique on a patient undergoing general anesthesia for dental restorations.
Medicine, Issue 47, difficult ventilation, difficult intubation, nasal, saturation
A Murine Model of Subarachnoid Hemorrhage
Institutions: University of Munich Medical Center.
In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.
Medicine, Issue 81, Nervous System Diseases, Subarachnoid hemorrhage (SAH), mouse model, filament perforation, intracranial pressure monitoring, blood distribution, surgical technique
A Simplified Technique for Producing an Ischemic Wound Model
Institutions: University of Louisville.
One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 °C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.
Medicine, Issue 63, Wound, ischemia, rabbit, minimally invasive, model, diabetes, physiology
Dual-phase Cone-beam Computed Tomography to See, Reach, and Treat Hepatocellular Carcinoma during Drug-eluting Beads Transarterial Chemo-embolization
Institutions: The Johns Hopkins Hospital, Philips Research North America, National Institutes of Health, Philips Healthcare.
The advent of cone-beam computed tomography (CBCT) in the angiography suite has been revolutionary in interventional radiology. CBCT offers 3 dimensional (3D) diagnostic imaging in the interventional suite and can enhance minimally-invasive therapy beyond the limitations of 2D angiography alone. The role of CBCT has been recognized in transarterial chemo-embolization (TACE) treatment of hepatocellular carcinoma (HCC). The recent introduction of a CBCT technique: dual-phase CBCT (DP-CBCT) improves intra-arterial HCC treatment with drug-eluting beads (DEB-TACE). DP-CBCT can be used to localize liver tumors with the diagnostic accuracy of multi-phasic multidetector computed tomography (M-MDCT) and contrast enhanced magnetic resonance imaging (CE-MRI) (See the tumor), to guide intra-arterially guidewire and microcatheter to the desired location for selective therapy (Reach the tumor), and to evaluate treatment success during the procedure (Treat the tumor). The purpose of this manuscript is to illustrate how DP-CBCT is used in DEB-TACE to see, reach, and treat HCC.
Medicine, Issue 82, Carcinoma, Hepatocellular, Tomography, X-Ray Computed, Surgical Procedures, Minimally Invasive, Digestive System Diseases, Diagnosis, Therapeutics, Surgical Procedures, Operative, Equipment and Supplies, Transarterial chemo-embolization, Hepatocellular carcinoma, Dual-phase cone-beam computed tomography, 3D roadmap, Drug-Eluting Beads
Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats
Institutions: Louisiana State University Health Science Center, Shreveport.
Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1
in 1992 and further characterized by Zhang et al.
. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.
Medicine, Issue 91, ischemic stroke, model, embolus, middle cerebral artery occlusion, thrombolytic therapy
Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves
Institutions: Florida International University, University of Florida , University of Florida , Jeddah, Saudi Arabia.
Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro
pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.
Bioengineering, Issue 80, Cardiovascular Diseases, Circulatory and Respiratory Physiological Phenomena, Fluid Mechanics and Thermodynamics, Mechanical Engineering, valve disease, valve replacement, polymer valves, pulse duplicator, modification, tri-leaflet geometries, hydrodynamic studies, relative assessment, medicine, bioengineering, physiology
Magnetic Resonance Imaging Quantification of Pulmonary Perfusion using Calibrated Arterial Spin Labeling
Institutions: University of California San Diego - UCSD, University of California San Diego - UCSD, University of California San Diego - UCSD.
This demonstrates a MR imaging method to measure the spatial distribution of pulmonary blood flow in healthy subjects
during normoxia (inspired O2
, fraction (FI
) = 0.21) hypoxia (FI
= 0.125), and hyperoxia
= 1.00). In addition, the physiological responses of the subject are monitored in the MR scan environment. MR images
were obtained on a 1.5 T GE MRI scanner during a breath hold from a sagittal slice in the right lung at functional residual capacity. An arterial
spin labeling sequence (ASL-FAIRER) was used to measure the spatial distribution of pulmonary blood flow 1,2
and a multi-echo fast
gradient echo (mGRE) sequence 3
was used to quantify the regional proton (i.e. H2
O) density, allowing the quantification
of density-normalized perfusion for each voxel (milliliters blood per minute per gram lung tissue).
With a pneumatic switching valve and facemask equipped with a 2-way non-rebreathing valve, different oxygen concentrations
were introduced to the subject in the MR scanner through the inspired gas tubing. A metabolic cart collected expiratory gas via expiratory tubing. Mixed expiratory O2
concentrations, oxygen consumption, carbon dioxide production, respiratory exchange ratio,
respiratory frequency and tidal volume were measured. Heart rate and oxygen saturation were monitored using pulse-oximetry.
Data obtained from a normal subject showed that, as expected, heart rate was higher in hypoxia (60 bpm) than during normoxia (51) or hyperoxia (50) and the arterial oxygen saturation (SpO2
) was reduced during hypoxia to 86%. Mean ventilation was 8.31 L/min BTPS during hypoxia, 7.04 L/min during normoxia, and 6.64 L/min during hyperoxia. Tidal volume was 0.76 L during hypoxia, 0.69 L during normoxia, and 0.67 L during hyperoxia.
Representative quantified ASL data showed that the mean density normalized perfusion was 8.86 ml/min/g during hypoxia, 8.26 ml/min/g during normoxia and 8.46 ml/min/g during hyperoxia, respectively. In this subject, the relative dispersion4
, an index of global heterogeneity, was increased in hypoxia (1.07 during hypoxia, 0.85 during normoxia, and 0.87 during hyperoxia) while the fractal dimension (Ds), another index of heterogeneity reflecting vascular branching structure, was unchanged (1.24 during hypoxia, 1.26 during normoxia, and 1.26 during hyperoxia).
Overview. This protocol will demonstrate the acquisition of data to measure the distribution of pulmonary perfusion noninvasively under conditions of normoxia, hypoxia, and hyperoxia using a magnetic resonance imaging technique known as arterial spin labeling (ASL).
Rationale: Measurement of pulmonary blood flow and lung proton density using MR technique offers high spatial resolution images which can be quantified and the ability to perform repeated measurements under several different physiological conditions. In human studies, PET, SPECT, and CT are commonly used as the alternative techniques. However, these techniques involve exposure to ionizing radiation, and thus are not suitable for repeated measurements in human subjects.
Medicine, Issue 51, arterial spin labeling, lung proton density, functional lung imaging, hypoxic pulmonary vasoconstriction, oxygen consumption, ventilation, magnetic resonance imaging
The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects
Institutions: University and Bern University Hospital (Inselspital), Kantonsspital Aarau, Boston Children's Hospital, Boston Children's Hospital, University and Bern University Hospital (Inselspital), University Hospital Cologne, Länggasse Bern.
Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.
Medicine, Issue 92,
Subarachnoid hemorrhage, animal models, rabbit, extracorporeal blood shunt, early brain injury, delayed cerebral vasospasm, microsurgery.
Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase
Institutions: Stanford University School of Medicine, Stanford University School of Medicine.
Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of abdominal aortic aneurysm (AAA) ever since it was first described in rats by Anidjar and colleagues.1
The rationale for its development was based on the disrupted nature of elastin observed in AAAs. This rat model has been modified to produce AAAs in the infrarenal aortic region of mice.2
The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice. Moreover, it is a viable platform to test potential therapeutic agents for AAA. In this video, we demonstrate the elastase infusion AAA procedure used in our laboratory.
Mice are anesthetized using 2.5% isoflurane, and a laparotomy is performed under sterile conditions. The abdominal aortais isolated with the assistance of an operating stereomicroscope (Leica). After placing temporary ligatures around the proximal and distal aorta, an aortotomy is created at the bifurcation with the tip of a 30-gauge needle. A heat-tapered segment of PE-10 polyethylene tubing is introduced through the aortotomy and secured. The aortic lumen is subsequently perfused for 5-15 minutes at 100 mm Hg with saline containing type I porcine pancreatic elastase (4.5 U/mL; Sigma Chemical Co.). After removing the perfusion catheter, the aortotomy is repaired without constriction of the lumen.
Medicine, Issue 29, abdominal aortic aneurysm, AAA, mouse, elastase
Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection
Institutions: Children's Hospital of Pittsburgh of UPMC, Tufts University Medical Center.
The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro
. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.
Cancer Biology, Issue 77, Cellular Biology, Molecular Biology, Medicine, Biochemistry, Biomedical Engineering, Acinar Cells, Pancreatitis, Transfection, Microscopy, Confocal, Calcium Signaling, Pancreatic Acinar Cells, Pancreatitis, Calcium Signaling, Cytotoxicity, LDH Leakage, cell injury, imaging
A New Murine Model of Endovascular Aortic Aneurysm Repair
Institutions: Hôpital X. Bichat, AP-HP, Paris, Institut Galilée - Université Paris 13, Paris, France, Université Paris-Est Creteil, Ecole de chirurgie de l'assistance publique des hôpitaux de Paris, Université René Descartes.
Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901
in a rat, then described in mice2
. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.
Medicine, Issue 77, Anatomy, Physiology, Biomedical Engineering, Bioengineering, Cardiology, Aortic Diseases, Aortic Aneurysm, Aortic Aneurysm, Disease Models, Animal, Vascular Surgical Procedures, Vascular Grafting, Microsurgery, animal models, Cardiovascular Diseases, Abdominal aortic aneurysm, rat, stentgraft exclusion, EVAR, animal model
Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System
Institutions: Case Western Reserve University School of Medicine, Cleveland, Case Western Reserve University School of Medicine, Cleveland, Case Western Reserve University School of Medicine, Cleveland.
The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).
Medicine, Issue 80, Crohn's disease, ulcerative colitis, colon cancer, Clostridium difficile, SAMP mice, DSS/AOM-colitis, decimal scoring identifier
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
Microsurgical Venous Pouch Arterial-Bifurcation Aneurysms in the Rabbit Model: Technical Aspects
Institutions: Hospital Rudolfstiftung, Kantonsspital Aarau, Medical University of Vienna, University of Berne, Medical University of Vienna, Paracelsus University Salzburg.
For ruptured human cerebral aneurysms endovascular embolization has become an equivalent alternative to aneurysm clipping.1
However, large clinical trials have shown disappointing long-term results with unacceptable high rates of aneurysm recanalization and delayed aneurysm rupture.2
To overcome these problems, animal experimental studies are crucial for the development of better endovascular devices.3-5
Several animal models in rats, rabbits, canines and swine are available.6-8
Comparisons of the different animal models showed the superiority of the rabbit model with regard to hemodynamics and comparability of the coagulation system and cost-effectiveness.9-11
The venous pouch arterial bifurcation model in rabbits is formed by a venous pouch sutured into an artificially created true bifurcation of both common carotid arteries (CCA). The main advantage of this model are true bifurcational hemodynamics.12
The major drawbacks are the sofar high microsurgical technical demands and high morbidity and mortality rates of up to 50%.13
These limitations have resulted in less frequent use of this aneurysm model in the recent years. These shortcomings could be overcome with improved surgical procedures and modified peri- and postoperative analgetic management and anticoagulation.14-16
Our techniques reported in this paper demonstrate this optimized technique for microsurgical creation of arterial bifurcation aneurysms.
Medicine, Issue 51, mental aneurysm, bifurcation, microsurgery, endovascular-coiling
Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers
Institutions: University of Maryland, University of Maryland.
Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200 nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125
I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125
I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.
Bioengineering, Issue 80, Antigens, Enzymes, Biological Therapy, bioengineering (general), Pharmaceutical Preparations, Macromolecular Substances, Therapeutics, Digestive System and Oral Physiological Phenomena, Biological Phenomena, Cell Physiological Phenomena, drug delivery systems, targeted nanocarriers, transcellular transport, epithelial cells, tight junctions, transepithelial electrical resistance, endocytosis, transcytosis, radioisotope tracing, immunostaining
Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation
Institutions: University Hospital Münster, University Children's Hospital Münster.
Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo,
necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live
mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo
murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo
imaging and may significantly impact on preclinical research in various fields.
Medicine, Issue 90,
gastroenterology, in vivo imaging, murine endoscopy, diagnostic imaging, carcinogenesis, intestinal wound healing, experimental colitis
High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres
Institutions: George Mason University, Virginia Surgery Associates.
Breast ductal carcinoma in situ
(DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2
incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.
Cancer Biology, Issue 93, Breast, ductal carcinoma in situ, epidermal growth factor, mammosphere, organoid, pre-invasive, primary cell culture, serum-free, spheroid
Measuring Attentional Biases for Threat in Children and Adults
Institutions: Rutgers University.
Investigators have long been interested in the human propensity for the rapid detection of threatening stimuli. However, until recently, research in this domain has focused almost exclusively on adult participants, completely ignoring the topic of threat detection over the course of development. One of the biggest reasons for the lack of developmental work in this area is likely the absence of a reliable paradigm that can measure perceptual biases for threat in children. To address this issue, we recently designed a modified visual search paradigm similar to the standard adult paradigm that is appropriate for studying threat detection in preschool-aged participants. Here we describe this new procedure. In the general paradigm, we present participants with matrices of color photographs, and ask them to find and touch a target on the screen. Latency to touch the target is recorded. Using a touch-screen monitor makes the procedure simple and easy, allowing us to collect data in participants ranging from 3 years of age to adults. Thus far, the paradigm has consistently shown that both adults and children detect threatening stimuli (e.g.,
snakes, spiders, angry/fearful faces) more quickly than neutral stimuli (e.g.,
flowers, mushrooms, happy/neutral faces). Altogether, this procedure provides an important new tool for researchers interested in studying the development of attentional biases for threat.
Behavior, Issue 92, Detection, threat, attention, attentional bias, anxiety, visual search
A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics
Institutions: Hospital for Sick Children, Toronto Medical Discovery Tower, Princess Margaret Hospital, Toronto Western Hospital.
We have successfully integrated previously established Intracranial window (ICW) technology 1-4
with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 μm from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7
. In addition, the ICW provides many challenges when optimizing the imaging.
Cancer Biology, Issue 76, Medicine, Biomedical Engineering, Cellular Biology, Molecular Biology, Genetics, Neuroscience, Neurobiology, Biophysics, Anatomy, Physiology, Surgery, Intracranial Window, In vivo imaging, Stereotactic radiation, Bone Marrow Derived Cells, confocal microscopy, two-photon microscopy, drug-cell interactions, drug kinetics, brain, imaging, tumors, animal model
Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase
Institutions: Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg.
HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies primarily target virus enzymes or virus-cell fusion, suppressing the viral life cycle without eradicating the infection. Since the integrated provirus is not targeted by these approaches, new resistant strains of HIV-1 may emerge. Here, we report that the engineered recombinase Tre (see Molecular evolution of the Tre recombinase , Buchholz, F., Max Planck Institute for Cell Biology and Genetics, Dresden) efficiently excises integrated HIV-1 proviral DNA from the genome of infected cells. We produced loxLTR containing viral pseudotypes and infected HeLa cells to examine whether Tre recombinase can excise the provirus from the genome of HIV-1 infected human cells. A virus particle-releasing cell line was cloned and transfected with a plasmid expressing Tre or with a parental control vector. Recombinase activity and virus production were monitored. All assays demonstrated the efficient deletion of the provirus from infected cells without visible cytotoxic effects. These results serve as proof of principle that it is possible to evolve a recombinase to specifically target an HIV-1 LTR and that this recombinase is capable of excising the HIV-1 provirus from the genome of HIV-1-infected human cells.
Before an engineered recombinase could enter the therapeutic arena, however, significant obstacles need to be overcome. Among the most critical issues, that we face, are an efficient and safe delivery to targeted cells and the absence of side effects.
Medicine, Issue 16, HIV, Cell Biology, Recombinase, provirus, HeLa Cells
Microsurgical Clip Obliteration of Middle Cerebral Aneurysm Using Intraoperative Flow Assessment
Institutions: Havard Medical School, Massachusetts General Hospital.
Cerebral aneurysms are abnormal widening or ballooning of a localized segment of an intracranial blood vessel. Surgical clipping is an important treatment for aneurysms which attempts to exclude blood from flowing into the aneurysmal segment of the vessel while preserving blood flow in a normal fashion. Improper clip placement may result in residual aneurysm with the potential for subsequent aneurysm rupture or partial or full occlusion of distal arteries resulting in cerebral infarction. Here we describe the use of an ultrasonic flow probe to provide quantitative evaluation of arterial flow before and after microsurgical clip placement at the base of a middle cerebral artery aneurysm. This information helps ensure adequate aneurysm reconstruction with preservation of normal distal blood flow.
Medicine, Issue 31, Aneurysm, intraoperative, brain, surgery, surgical clipping, blood flow, aneurysmal segment, ultrasonic flow probe