Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.
22 Related JoVE Articles!
Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers
Institutions: The Ohio State University Wexner Medical Center, The Ohio State University Wexner Medical Center, Rush University Medical Center, The Ohio State University Wexner Medical Center.
Maintaining homeostatic Ca2+
signaling is a fundamental physiological process in living cells. Ca2+
sparks are the elementary units of Ca2+
signaling in the striated muscle fibers that appear as highly localized Ca2+
release events mediated by ryanodine receptor (RyR) Ca2+
release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+
sparks could provide information on the intracellular Ca2+
handling properties of healthy and diseased striated muscles. Although Ca2+
sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+
sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+
indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+
sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+
sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3
R) and RyR contributes to Ca2+
spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+
sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx
mice) or amyotrophic lateral sclerosis (ALS model).
Physiology, Issue 84, flexor digitorm brevis (FDB), sarcoplasmic reticulum, SR Ca2+ release, calcium signaling, ryanodine receptor, confocal imaging, muscle physiology
The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern
Institutions: University of Cologne.
Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of abdominal ganglia (A1 to A6). This chain of ganglia includes the part of the central nervous system (CNS) that drives coordinated locomotion of the pleopods (swimmerets): the swimmeret system. It is known for over five decades that in crayfish each swimmeret is driven by its own independent pattern generating kernel that generates rhythmic alternating activity 1-3
. The motor neurons innervating the musculature of each swimmeret comprise two anatomically and functionally distinct populations 4
. One is responsible for the retraction (power stroke, PS) of the swimmeret. The other drives the protraction (return stroke, RS) of the swimmeret. Motor neurons of the swimmeret system are able to produce spontaneously a fictive motor pattern, which is identical to the pattern recorded in vivo 1
The aim of this report is to introduce an interesting and convenient model system for studying rhythm generating networks and coordination of independent microcircuits for students’ practical laboratory courses. The protocol provided includes step-by-step instructions for the dissection of the crayfish’s abdominal nerve cord, pinning of the isolated chain of ganglia, desheathing the ganglia and recording the swimmerets fictive motor pattern extracellularly from the isolated nervous system.
Additionally, we can monitor the activity of swimmeret neurons recorded intracellularly from dendrites. Here we also describe briefly these techniques and provide some examples. Furthermore, the morphology of swimmeret neurons can be assessed using various staining techniques. Here we provide examples of intracellular (by iontophoresis) dye filled neurons and backfills of pools of swimmeret motor neurons. In our lab we use this preparation to study basic functions of fictive locomotion, the effect of sensory feedback on the activity of the CNS, and coordination between microcircuits on a cellular level.
Neurobiology, Issue 93, crustacean, dissection, extracellular recording, fictive locomotion, motor neurons, locomotion
In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis
Institutions: University Hospital of Strasbourg, University of Strasbourg, University Hospital of Strasbourg.
Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo
macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo
noninvasive evaluation of macrophages infiltration and an assessment of therapy action.
Medicine, Issue 80, Biomedical Engineering, Anatomy, Physiology, Molecular Biology, Diagnostic Imaging, Musculoskeletal System, Bacterial Infections and Mycoses, Macrophage, MR imaging, infection, arthritis, USPIO, imaging, clinical techniques
Diffusion Tensor Magnetic Resonance Imaging in the Analysis of Neurodegenerative Diseases
Institutions: University of Ulm.
Diffusion tensor imaging (DTI) techniques provide information on the microstructural processes of the cerebral white matter (WM) in vivo
. The present applications are designed to investigate differences of WM involvement patterns in different brain diseases, especially neurodegenerative disorders, by use of different DTI analyses in comparison with matched controls.
DTI data analysis is performed in a variate fashion, i.e.
voxelwise comparison of regional diffusion direction-based metrics such as fractional anisotropy (FA), together with fiber tracking (FT) accompanied by tractwise fractional anisotropy statistics (TFAS) at the group level in order to identify differences in FA along WM structures, aiming at the definition of regional patterns of WM alterations at the group level. Transformation into a stereotaxic standard space is a prerequisite for group studies and requires thorough data processing to preserve directional inter-dependencies. The present applications show optimized technical approaches for this preservation of quantitative and directional information during spatial normalization in data analyses at the group level. On this basis, FT techniques can be applied to group averaged data in order to quantify metrics information as defined by FT. Additionally, application of DTI methods, i.e.
differences in FA-maps after stereotaxic alignment, in a longitudinal analysis at an individual subject basis reveal information about the progression of neurological disorders. Further quality improvement of DTI based results can be obtained during preprocessing by application of a controlled elimination of gradient directions with high noise levels.
In summary, DTI is used to define a distinct WM pathoanatomy of different brain diseases by the combination of whole brain-based and tract-based DTI analysis.
Medicine, Issue 77, Neuroscience, Neurobiology, Molecular Biology, Biomedical Engineering, Anatomy, Physiology, Neurodegenerative Diseases, nuclear magnetic resonance, NMR, MR, MRI, diffusion tensor imaging, fiber tracking, group level comparison, neurodegenerative diseases, brain, imaging, clinical techniques
Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration
Institutions: University College London, San Raffaele Hospital.
Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g.
lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro
with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo
, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.
Bioengineering, Issue 83, Skeletal Muscle, Muscle Cells, Muscle Fibers, Skeletal, Pericytes, Stem Cells, Induced Pluripotent Stem Cells (iPSCs), Muscular Dystrophies, Cell Differentiation, animal models, muscle stem/progenitor cells, mesoangioblasts, muscle regeneration, iPSC-derived mesoangioblasts (IDEMs)
Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice
Institutions: St Michaels Hospital, McMaster University.
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.
Medicine, Issue 81, mouse, tibial nerve, gastronemius, soleus, atrophy, denervation, reinnervation, myofiber, transection
Oscillation and Reaction Board Techniques for Estimating Inertial Properties of a Below-knee Prosthesis
Institutions: University of Northern Colorado, Arizona State University, Iowa State University.
The purpose of this study was two-fold: 1) demonstrate a technique that can be used to directly estimate the inertial properties of a below-knee prosthesis, and 2) contrast the effects of the proposed technique and that of using intact limb inertial properties on joint kinetic estimates during walking in unilateral, transtibial amputees. An oscillation and reaction board system was validated and shown to be reliable when measuring inertial properties of known geometrical solids. When direct measurements of inertial properties of the prosthesis were used in inverse dynamics modeling of the lower extremity compared with inertial estimates based on an intact shank and foot, joint kinetics at the hip and knee were significantly lower during the swing phase of walking. Differences in joint kinetics during stance, however, were smaller than those observed during swing. Therefore, researchers focusing on the swing phase of walking should consider the impact of prosthesis inertia property estimates on study outcomes. For stance, either one of the two inertial models investigated in our study would likely lead to similar outcomes with an inverse dynamics assessment.
Bioengineering, Issue 87, prosthesis inertia, amputee locomotion, below-knee prosthesis, transtibial amputee
Utility of Dissociated Intrinsic Hand Muscle Atrophy in the Diagnosis of Amyotrophic Lateral Sclerosis
Institutions: Westmead Hospital, University of Sydney, Australia.
The split hand
phenomenon refers to predominant wasting of thenar muscles and is an early and specific feature of amyotrophic lateral sclerosis (ALS). A novel split hand index (SI) was developed to quantify the split hand phenomenon, and its diagnostic utility was assessed in ALS patients. The split hand index was derived by dividing the product of the compound muscle action potential (CMAP) amplitude recorded over the abductor pollicis brevis and first dorsal interosseous muscles by the CMAP amplitude recorded over the abductor digiti minimi muscle. In order to assess the diagnostic utility of the split hand index, ALS patients were prospectively assessed and their results were compared to neuromuscular disorder patients. The split hand index was significantly reduced in ALS when compared to neuromuscular disorder patients (P<0.0001). Limb-onset ALS patients exhibited the greatest reduction in the split hand index, and a value of 5.2 or less reliably differentiated ALS from other neuromuscular disorders. Consequently, the split hand index appears to be a novel diagnostic biomarker for ALS, perhaps facilitating an earlier diagnosis.
Medicine, Issue 85, Amyotrophic Lateral Sclerosis (ALS), dissociated muscle atrophy, hypothenar muscles, motor neuron disease, split-hand index, thenar muscles
Transcranial Magnetic Stimulation for Investigating Causal Brain-behavioral Relationships and their Time Course
Institutions: University College London.
Transcranial magnetic stimulation (TMS) is a safe, non-invasive brain stimulation technique that uses a strong electromagnet in order to temporarily disrupt information processing in a brain region, generating a short-lived “virtual lesion.” Stimulation that interferes with task performance indicates that the affected brain region is necessary to perform the task normally. In other words, unlike neuroimaging methods such as functional magnetic resonance imaging (fMRI) that indicate correlations between brain and behavior, TMS can be used to demonstrate causal brain-behavior relations. Furthermore, by varying the duration and onset of the virtual lesion, TMS can also reveal the time course of normal processing. As a result, TMS has become an important tool in cognitive neuroscience. Advantages of the technique over lesion-deficit studies include better spatial-temporal precision of the disruption effect, the ability to use participants as their own control subjects, and the accessibility of participants. Limitations include concurrent auditory and somatosensory stimulation that may influence task performance, limited access to structures more than a few centimeters from the surface of the scalp, and the relatively large space of free parameters that need to be optimized in order for the experiment to work. Experimental designs that give careful consideration to appropriate control conditions help to address these concerns. This article illustrates these issues with TMS results that investigate the spatial and temporal contributions of the left supramarginal gyrus (SMG) to reading.
Behavior, Issue 89,
Transcranial magnetic stimulation, virtual lesion, chronometric, cognition, brain, behavior
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
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
Molecular Imaging to Target Transplanted Muscle Progenitor Cells
Institutions: Lawson Health Research Institute, Western University, Western University.
Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2
. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4
. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc
], monomeric red fluorescent protein [mrfp
] and sr39 thymidine kinase [sr39tk
]) whose expression can be imaged with different imaging modalities9, 10
. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11
. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc
) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14
. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11
, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.
Medicine, Issue 73, Medicine, Biophysics, Biomedical Engineering, Cellular Biology, Anatomy, Physiology, Genetics, Surgery, Diseases, Musculoskeletal Diseases, Analytical, Diagnostic and Therapeutic Techniques and Equipment, Therapeutics, Bioluminescence imaging (BLI), Reporter Gene Expression, Non-invasive Targeting, Muscle Progenitor Cells, Myoblasts, transplantation, cell implantation, MRI, PET, SPECT, BLI, imaging, clinical techniques, animal model
Pseudofracture: An Acute Peripheral Tissue Trauma Model
Institutions: University of Pittsburgh, University of Aachen Medical Center.
Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; this response is often accompanied by a delayed immunosuppression that adds the clinical complications of infection and can also increase mortality.1-9
Many studies have begun to assess these changes in the reactivity of the immune system following trauma.10-15
Immunologic studies are greatly supported through the wide variety of transgenic and knockout mice available for in vivo
modeling; these strains aid in detailed investigations to assess the molecular pathways involved in the immunologic responses.16-21
The challenge in experimental murine trauma modeling is long term investigation, as fracture fixation techniques in mice, can be complex and not easily reproducible.22-30
This pseudofracture model, an easily reproduced trauma model, overcomes these difficulties by immunologically mimicking an extremity fracture environment, while allowing freedom of movement in the animals and long term survival without the continual, prolonged use of anaesthesia. The intent is to recreate the features of long bone fracture; injured muscle and soft tissue are exposed to damaged bone and bone marrow without breaking the native bone.
The pseudofracture model consists of two parts: a bilateral muscle crush injury to the hindlimbs, followed by injection of a bone solution into these injured muscles. The bone solution is prepared by harvesting the long bones from both hindlimbs of an age- and weight-matched syngeneic donor. These bones are then crushed and resuspended in phosphate buffered saline to create the bone solution.
Bilateral femur fracture is a commonly used and well-established model of extremity trauma, and was the comparative model during the development of the pseudofracture model. Among the variety of available fracture models, we chose to use a closed method of fracture with soft tissue injury as our comparison to the pseudofracture, as we wanted a sterile yet proportionally severe peripheral tissue trauma model. 31
Hemorrhagic shock is a common finding in the setting of severe trauma, and the global hypoperfusion adds a very relevant element to a trauma model. 32-36
The pseudofracture model can be easily combined with a hemorrhagic shock model for a multiple trauma model of high severity. 37
Medicine, Issue 50, Trauma, musculoskeletal, mouse, extremity, inflammation, immunosuppression, immune response.
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
Quantitative Autonomic Testing
Institutions: University of Massachusetts Medical School.
Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be invaluable tool for evaluation of these disorders, both in clinic and research. There are number of autonomic tests, however, only few were validated clinically or are quantitative. Here, fully quantitative and clinically validated protocol for testing of autonomic functions is presented. As a bare minimum the clinical autonomic laboratory should have a tilt table, ECG monitor, continuous noninvasive blood pressure monitor, respiratory monitor and a mean for evaluation of sudomotor domain. The software for recording and evaluation of autonomic tests is critical for correct evaluation of data. The presented protocol evaluates 3 major autonomic domains: cardiovagal, adrenergic and sudomotor. The tests include deep breathing, Valsalva maneuver, head-up tilt, and quantitative sudomotor axon test (QSART). The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores (CASS). Detailed protocol is provided highlighting essential aspects of testing with emphasis on proper data acquisition, obtaining the relevant parameters and unbiased evaluation of autonomic signals. The normative data and CASS algorithm for interpretation of results are provided as well.
Medicine, Issue 53, Deep breathing, Valsalva maneuver, tilt test, sudomotor testing, Composite Autonomic Severity Score, CASS
Thermal Ablation for the Treatment of Abdominal Tumors
Institutions: University of Wisconsin-Madison, University of Wisconsin-Madison.
Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ablation procedure, a thin applicator is guided into the target tumor under imaging guidance. Energy is then applied to the tissue until temperatures rise to cytotoxic levels (50-60 °C). Various energy sources are available to heat biological tissues, including radiofrequency (RF) electrical current, microwaves, laser light and ultrasonic waves. Of these, RF and microwave ablation are most commonly used worldwide.
During RF ablation, alternating electrical current (~500 kHz) produces resistive heating around the interstitial electrode. Skin surface electrodes (ground pads) are used to complete the electrical circuit. RF ablation has been in use for nearly 20 years, with good results for local tumor control, extended survival and low complication rates1,2
. Recent studies suggest RF ablation may be a first-line treatment option for small hepatocellular carcinoma and renal-cell carcinoma3-5
. However, RF heating is hampered by local blood flow and high electrical impedance tissues (eg, lung, bone, desiccated or charred tissue)6,7
. Microwaves may alleviate some of these problems by producing faster, volumetric heating8-10
. To create larger or conformal ablations, multiple microwave antennas can be used simultaneously while RF electrodes require sequential operation, which limits their efficiency. Early experiences with microwave systems suggest efficacy and safety similar to, or better than RF devices11-13
Alternatively, cryoablation freezes the target tissues to lethal levels (-20 to -40 °C). Percutaneous cryoablation has been shown to be effective against RCC and many metastatic tumors, particularly colorectal cancer, in the liver14-16
. Cryoablation may also be associated with less post-procedure pain and faster recovery for some indications17
. Cryoablation is often contraindicated for primary liver cancer due to underlying coagulopathy and associated bleeding risks frequently seen in cirrhotic patients. In addition, sudden release of tumor cellular contents when the frozen tissue thaws can lead to a potentially serious condition known as cryoshock 16
Thermal tumor ablation can be performed at open surgery, laparoscopy or using a percutaneous approach. When performed percutaneously, the ablation procedure relies on imaging for diagnosis, planning, applicator guidance, treatment monitoring and follow-up. Ultrasound is the most popular modality for guidance and treatment monitoring worldwide, but computed tomography (CT) and magnetic resonance imaging (MRI) are commonly used as well. Contrast-enhanced CT or MRI are typically employed for diagnosis and follow-up imaging.
Medicine, Issue 49, Thermal ablation, interventional oncology, image-guided therapy, radiology, cancer
Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems
Institutions: Robert Wood Johnson Medical School .
Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo
function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo
membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.
Cell Biology, Issue 52, mouse, cell membrane, muscle injury, tissue repair, treadmill, MG53, confocal microscopy, vesicle trafficking
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
Breathing-controlled Electrical Stimulation (BreEStim) for Management of Neuropathic Pain and Spasticity
Institutions: University of Texas Health Science Center at Houston , TIRR Memorial Hermann Hospital, TIRR Memorial Hermann Hospital.
Electrical stimulation (EStim) refers to the application of electrical current to muscles or nerves in order to achieve functional and therapeutic goals. It has been extensively used in various clinical settings. Based upon recent discoveries related to the systemic effects of voluntary breathing and intrinsic physiological interactions among systems during voluntary breathing, a new EStim protocol, Breathing-controlled Electrical Stimulation (BreEStim), has been developed to augment the effects of electrical stimulation. In BreEStim, a single-pulse electrical stimulus is triggered and delivered to the target area when the airflow rate of an isolated voluntary inspiration reaches the threshold. BreEStim integrates intrinsic physiological interactions that are activated during voluntary breathing and has demonstrated excellent clinical efficacy. Two representative applications of BreEStim are reported with detailed protocols: management of post-stroke finger flexor spasticity and neuropathic pain in spinal cord injury.
Medicine, Issue 71, Neuroscience, Neurobiology, Anatomy, Physiology, Behavior, electrical stimulation, BreEStim, electrode, voluntary breathing, respiration, inspiration, pain, neuropathic pain, pain management, spasticity, stroke, spinal cord injury, brain, central nervous system, CNS, clinical, electromyogram, neuromuscular electrical stimulation
DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation
Institutions: David Geffen School of Medicine, University of California, Los Angeles.
A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo
transfection of plasmids by various methods into specific tissues such as liver1
, skeletal muscle2,3
, and even the brain4
. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis
(FDB) and interosseus
(IO) muscles of adult mice using an in vivo
electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3
, calpain, FKBP12, β2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and α-actinin; and membrane proteins, i.e. α1s-DHPR, RyR1, cardiac Na/Ca2+
exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3
and functional evidence5
. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8
Cellular Biology, Issue 32, electroporation, skeletal muscle, plasmids, protein expression, mouse, two-photon microscopy, fluorescence, transgenic
Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain
Institutions: University of Calgary.
Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis. In some women the severe episodes of cyclic pain change and the resultant pain becomes continuous and this condition becomes known as Chronic Pelvic Pain. This state can be present even after the appropriate medical or surgical therapy has been instituted. It can be associated with pain and tenderness in the muscles of the abdomen wall and intra-pelvic muscles leading to severe dyspareunia. Additional symptoms of irritable bowel and interstitial cystitis are common. A common sign of the development of this state is the emergence of cutaneous allodynia
which emerges from the so-called viscero-somatic reflex. A simple bedside test for the presence of cutaneous allodynia
is presented that does not require excessive time or special equipment. This test builds on previous work associated with changes in sensation related to gall bladder function and the viscera-somatic reflex(1;2).
The test is undertaken with the subject s permission after an explanation of how the test will be performed. Allodynia
refers to a condition in which a stimulus that is not normally painful is interpreted by the subject as painful. In this instance the light touch associated with a cotton-tipped applicator would not be expected to be painful. A positive test is however noted by the woman as suddenly painful or suddenly sharp. The patterns of this sensation are usually in a discrete pattern of a dermatome of the nerves that innervate the pelvis.
The underlying pathology is now interpreted as evidence of neuroplasticity as a consequence of severe and repeating pain with changes in the functions of the dorsal horns of the spinal cord that results in altered function of visceral tissues and resultant somatic symptoms(3).
The importance of recognizing the condition lies in an awareness that this process may present coincidentally with the initiating condition or after it has been treated. It also permits the clinician to evaluate the situation from the perspective that alternative explanations for the pain may be present that may not require additional surgery.
Medicine, Issue 28, Chronic pelvic pain, cutaneous allodynia, trigger points, dysmenorrhea, endometriosis, dyspareunia
Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia
Institutions: Stanford University , Stanford University .
Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1
Medicine, Issue 23, hindlimb ischemia, peripheral arterial disease, embryonic stem cell, cell transplantation, bioluminescence imaging, non-invasive tracking, mouse model