The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.
16 Related JoVE Articles!
Procedures for Rat in situ Skeletal Muscle Contractile Properties
Institutions: University of Calgary .
There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37°C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.
Physiology, Issue 56, physiological preparation, contractile properties, force-frequency relationship, force-length relationship
Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo
Institutions: Northwestern University Feinberg School of Medicine.
The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo
, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo
therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1
): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e.
the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro
preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4
, including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo
in adult mice5,6
. However, these recordings were obtained in paralyzed animals, i.e.
without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9
, or reproducting human disease10,11
, we expect this technique to be an essential tool for the study of spinal motor system.
Neuroscience, Issue 70, Physiology, Biophysics, Anatomy, Medicine, Motor System, Spinal Cord, Intracellular Recordings, Motoneurons, EMG, Force, lumbar, neuron, brain, mouse, animal model
Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies
Institutions: Université de Sherbrooke.
Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.
bioengineering, Issue 41, Rat tail tendon, extraction, cross-section, optic micrometer, anchors, bioreactor, biomechanics, mechanobiology
Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle
Institutions: Ottawa Hospital Research Institute, University of Ottawa .
Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro
system in which the physical association between the myofiber and its stem cells is preserved 1
. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3
. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6
. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7
. Although the myofiber culture system cannot substitute for in vivo
studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9
so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3
. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo
regenerative process 1-3
. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).
Stem Cell Biology, Issue 73, Cellular Biology, Molecular Biology, Medicine, Biomedical Engineering, Bioengineering, Physiology, Anatomy, Tissue Engineering, Stem Cells, Myoblasts, Skeletal, Satellite Cells, Skeletal Muscle, Muscular Dystrophy, Duchenne, Tissue Culture Techniques, Muscle regeneration, Pax7, isolation and culture of isolated myofibers, muscles, myofiber, immunostaining, cell culture, hindlimb, mouse, animal model
Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises
Institutions: University of Kentucky, University of Toronto.
The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+
on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.
Neuroscience, Issue 47, Invertebrate, Crayfish, neurophysiology, muscle, anatomy, electrophysiology
Reverse Total Shoulder Arthroplasty
Institutions: Case Western Reserve University.
Reverse total shoulder arthroplasty was initially approved for use in rotator cuff arthropathy and well as chronic pseudoparalysis without arthritis in patients who were not appropriate for tendon transfer reconstructions. Traditional surgical options for these patients were limited and functional results were sub-optimal and at times catastrophic. The use of reverse shoulder arthroplasty has been found to effectively restore these patients function and relieve symptoms associated with their disease. The procedure can be done through two approaches, the deltopectoral or the superolateral. Complication rates associated with the use of the prosthesis have ranged from 8-60% with more recent reports trending lower as experienced is gained. Salvage options for a failed reverse shoulder prosthesis are limited and often have significant associated disability. Indications for the use of this prosthesis continue to be evaluated including its use for revision arthroplasty, proximal humeral fracture and tumor. Careful patient selection is essential because of the significant risks associated with the procedure.
Medicine, Issue 53, Reverse, Total, Shoulder, Arthroplasty, Rotator Cuff, Arthropathy, Arthritis, Glenoid, Humerus, Fracture
Extraction of the EPP Component from the Surface EMG
Institutions: Matsumoto Dental University.
A surface electromyogram (EMG), especially when recorded near the neuromuscular junction, is expected to contain the endplate potential (EPP) component which can be extracted with an appropriate signal filter. Two factors are important: the EMG must be recorded in monopolar fashion, and the recording must be done so the low frequency signal corresponding the EPP is not eliminated. This report explains how to extract the EPP component from the EMG of the masseter muscle in a human subject. The surface EMG is recorded from eight sites using traditional disc electrodes aligned along over the muscle, with equal inter-electrode distance from the zygomatic arch to the angle of mandible in response to quick gum clenching. A reference electrode is placed on the tip of the nose. The EPP component is extracted from the raw EMGs by applying a high-cut digital filter (2nd dimension Butterworth filter) with a range of 10-35 Hz. When the filter is set to 10 Hz, the extracted EPP wave deflects either negative or positive depending on the recording site. The difference in the polarity reflects the sink-source relation of the end plate current, with the site showing the most negative deflection corresponding to the neuromuscular junction. In the case of the masseter muscle, the neuromuscular junction is estimated to be located in the inferior portion close to the angle of mandible. The EPP component exhibits an interesting oscillation when the cut-off frequency of the high-cut digital filter is set to 30 Hz. The EPP oscillation indicates that muscle contraction is adjusted in an intermittent manner. Abnormal tremors accompanying various sorts of diseases may be substantially due to this EPP oscillation, which becomes slower and is difficult to cease.
Neuroscience, Issue 34, masseter muscle, EMG, EPP, neuromuscular junction, EPP oscillation
Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles
Institutions: UMDNJ-Robert Wood Johnson Medical School, University of Missouri-Kansas City, Ohio State University .
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+
handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+
signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e.
, mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro
muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.
Physiology, Issue 69, extensor digitorum longus, soleus, in vitro contractility, calcium signaling, muscle-tendon complex, mechanic alternans
Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia
Institutions: Technische Universität München, University Hospital of Basel, University of Saarland, University Hospital Zurich.
Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al.
have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.
Medicine, Issue 93, flap, ischemia, microcirculation, angiogenesis, skin, necrosis, inflammation, apoptosis, preconditioning, persistent ischemia, in vivo model, muscle.
Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice
Institutions: University of Missouri.
Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2
. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3
. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4
. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5
. This later change increases muscle stiffness 6
. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.
Medicine, Issue 72, Immunology, Microbiology, Anatomy, Physiology, Molecular Biology, Muscle, Skeletal, Neuromuscular Diseases, Drug Therapy, Gene Therapy, Musculoskeletal Diseases, Skeletal Muscle, Tibialis Anterior, Contractile Properties, Passive Properties, EDL, TA, animal model
In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury
Institutions: Royal Infirmary of Edinburgh, Royal Infirmary of Edinburgh.
Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite tissue. Ischemia reperfusion injury (IRI) is a known cause of partial free flap failure and has no effective treatment. Establishing a laboratory model of this injury can prove costly both financially as larger mammals are conventionally used and in the expertise required by the technical difficulty of these procedures typically requires employing an experienced microsurgeon. This publication and video demonstrate the effective use of a model of IRI in rats which does not require microsurgical expertise. This procedure is an in situ
model of a transverse abdominis myocutaneous (TRAM) flap where atraumatic clamps are utilized to reproduce the ischemia-reperfusion injury associated with this surgery. A laser Doppler Imaging (LDI) scanner is employed to assess flap perfusion and the image processing software, Image J to assess percentage area skin survival as a primary outcome measure of injury.
Medicine, Issue 76, Biomedical Engineering, Immunology, Anatomy, Physiology, Cellular Biology, Hematology, Surgery, Microsurgery, Reconstructive Surgical Procedures, Surgical Procedures, Operative, Myocutaneous flap, preconditioning, ischemia reperfusion injury, rat, animal model
An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents
Institutions: San José State University, University of Florida, Emory University School of Medicine, San José State University.
Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro
study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro
preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.
Neuroscience, Issue 91, muscle spindle, muscle afferent, extensor digitorum longus, sensory neurons, electrophysiology
Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction
Institutions: RWTH-Aachen University, Kyoto University .
Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2
, immunological responses 3,4
, hemodynamics 5,6
, and small-for-size syndrome 7
. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8
since the first model was described by Lee et al. 9
in 1973, the elimination of the hepatic arterial reconstruction 10
and the introduction of the cuff anastomosis technique by Kamada et al. 11
were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al.
, the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12
and the liver integrity 8,13,14
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo
liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.
Medicine, Issue 73, Biomedical Engineering, Anatomy, Physiology, Immunology, Surgery, liver transplantation, liver, hepatic, partial, orthotopic, split, rat, graft, transplantation, microsurgery, procedure, clinical, technique, artery, arterialization, arterialized, anastomosis, reperfusion, rat, animal model
Ex vivo Mechanical Loading of Tendon
Institutions: University of California, Berkeley , University of California, San Francisco.
Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37°C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.
Developmental biology, issue 4, tendon, tension
Molecular Evolution of the Tre Recombinase
Institutions: Max Plank Institute for Molecular Cell Biology and Genetics, Dresden.
Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells.
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.
Cell Biology, Issue 15, HIV-1, Tre recombinase, Site-specific recombination, molecular evolution
Laparoscopic Left Liver Sectoriectomy of Caroli's Disease Limited to Segment II and III
Institutions: University of Insubria, University of Insubria.
Caroli's disease is defined as a abnormal dilatation of the intra-hepatica bile ducts: Its incidence is extremely low (1 in 1,000,000 population) and in most of the cases the whole liver is interested and liver transplantation is the treatment of choice. In case of dilatation limited to the left or right lobe, liver resection can be performed. For many year the standard approach for liver resection has been a formal laparotomy by means of a large incision of abdomen that is characterized by significant post-operatie morbidity. More recently, minimally invasive, laparoscopic approach has been proposed as possible surgical technique for liver resection both for benign and malignant diseases. The main benefits of the minimally invasive approach is represented by a significant reduction of the surgical trauma that allows a faster recovery a less post-operative complications.
This video shows a case of Caroli s disease occured in a 58 years old male admitted at the gastroenterology department for sudden onset of abdominal pain associated with fever (>38C° ), nausea and shivering. Abdominal ultrasound demonstrated a significant dilatation of intra-hepatic left sited bile ducts with no evidences of gallbladder or common bile duct stones. Such findings were confirmed abdominal high resolution computer tomography.
Laparoscopic left sectoriectomy was planned. Five trocars and 30° optic was used, exploration of the abdominal cavity showed no adhesions or evidences of other diseases.
In order to control blood inflow to the liver, vascular clamp was placed on the hepatic pedicle (Pringle s manouvre), Parenchymal division is carried out with a combined use of 5 mm bipolar forceps and 5 mm ultrasonic dissector. A severely dilated left hepatic duct was isolated and divided using a 45mm endoscopic vascular stapler. Liver dissection was continued up to isolation of the main left portal branch that was then divided with a further cartridge of 45 mm vascular stapler.
At his point the left liver remains attached only by the left hepatic vein: division of the triangular ligament was performed using monopolar hook and the hepatic vein isolated and the divided using vascular stapler.
Haemostatis was refined by application of argon beam coagulation and no bleeding was revealed even after removal of the vascular clamp (total Pringle s time 27 minutes).
Postoperative course was uneventful, minimal elevation of the liver function tests was recorded in post-operative day 1 but returned to normal at discharged on post-operative day 3.
Medicine, Issue 24, Laparoscopy, Liver resection, Caroli's disease, Left sectoriectomy