The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.
24 Related JoVE Articles!
Dorsal Column Steerability with Dual Parallel Leads using Dedicated Power Sources: A Computational Model
In spinal cord stimulation (SCS), concordance of stimulation-induced paresthesia over painful body regions is a necessary condition for therapeutic efficacy. Since patient pain patterns can be unique, a common stimulation configuration is the placement of two leads in parallel in the dorsal epidural space. This construct provides flexibility in steering stimulation current mediolaterally over the dorsal column to achieve better pain-paresthesia overlap. Using a mathematical model with an accurate fiber diameter distribution, we studied the ability of dual parallel leads to steer stimulation between adjacent contacts on dual parallel leads using (1) a single source system, and (2) a multi-source system, with a dedicated current source for each contact. The volume conductor model of a low-thoracic spinal cord with epidurally-positioned dual parallel (2 mm separation) percutaneous leads was first created, and the electric field was calculated using ANSYS, a finite element modeling tool. The activating function for 10 um fibers was computed as the second difference of the extracellular potential along the nodes of Ranvier on the nerve fibers in the dorsal column. The volume of activation (VOA) and the central point of the VOA were computed using a predetermined threshold of the activating function. The model compared the field steering results with single source versus dedicated power source systems on dual 8-contact stimulation leads. The model predicted that the multi-source system can target more central points of stimulation on the dorsal column than a single source system (100 vs. 3) and the mean steering step for mediolateral steering is 0.02 mm for multi-source systems vs 1 mm for single source systems, a 50-fold improvement. The ability to center stimulation regions in the dorsal column with high resolution may allow for better optimization of paresthesia-pain overlap in patients.
Medicine, Issue 48, spinal cord stimulation, dorsal columns, current steering, field steering
Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders
Institutions: University of Trento, CNR Neuroscience Institute, Pisa, Italy.
Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-
) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/-
mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/-
mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.
Neuroscience, Issue 99, Immunohistochemistry, Morris water maze, ERK, hippocampus, cognition, memory, autism
A Novel Vertebral Stabilization Method for Producing Contusive Spinal Cord Injury
Institutions: Indiana University School of Medicine, Indiana University School of Medicine, Indiana University School of Medicine, Norton Healthcare.
Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing reliable cervical SCI is difficult due to lack of satisfactory methods of vertebral stabilization. The conventional method to stabilize the spine is to suspend the rostral and caudal cervical spine via clamps attached to cervical spinous processes. However, this method of stabilization fails to prevent tissue yielding during the contusion as the cervical spinal processes are too short to be effectively secured by the clamps (Figure 1
). Here we introduce a new method to completely stabilize the cervical vertebra at the same level of the impact injury. This method effectively minimizes movement of the spinal column at the site of impact, which greatly improves the production of consistent SCIs. We provide visual description of the equipment (Figure 2-4
), methods, and a step-by-step protocol for the stabilization of the cervical 5 vertebra (C5) of adult rats, to perform laminectomy (Figure 5
) and produce a contusive SCI thereafter. Although we only demonstrate a cervical hemi-contusion using the NYU/MASCIS impactor device, this vertebral stabilization technique can be applied to other regions of the spinal cord, or be adapted to other SCI devices. Improving spinal cord exposure and fixation through vertebral stabilization may be valuable for producing consistent and reliable injuries to the spinal cord. This vertebral stabilization method can also be used for stereotactic injections of cells and tracers, and for imaging using two-photon microscopy in various neurobiological studies.
Medicine, Issue 95, spine, vertebra, vertebrae, spinal cord injury, model, stabilization
A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells
Institutions: University of Basel.
Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1
. These limitations hamper the in vitro
study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2
. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo
neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3
, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.
Neuroscience, Issue 62, Human primary muscle cells, embryonic spinal cord explants, neurites, innervation, contraction, cell culture
Calibrated Forceps Model of Spinal Cord Compression Injury
Institutions: University of California, Davis, Shriners Hospitals for Children (Northern California).
Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al.
(2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.
Medicine, Issue 98, SCI, compression model, compression injury, modified forceps, laminectomy, neurological deficit, murine spinal cord, reproducible animal model, reproducible deficit
Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells
Institutions: University of Birmingham.
Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo
transplantation. In vivo
transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.
Cellular Biology, Issue 43, Skeletal muscle stem cell, embryonic tissue culture, apoptosis, growth factor, proliferation, myoblast, myogenesis, satellite cell, skeletal muscle differentiation, muscular dystrophy
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)
A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity
Institutions: Université de Versailles Saint-Quentin-en-Yvelines, Hôpital Ambroise Paré, Université de Versailles Saint-Quentin-en-Yvelines.
A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.
Physiology, Issue 87, rat, cervical spinal cord injury, respiratory deficit, crossed phrenic phenomenon, respiratory neuroplasticity
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Assessing Functional Performance in the Mdx Mouse Model
Institutions: Leiden University Medical Center.
Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential pharmaceutical compounds and gene therapy approaches have progressed into clinical trials. With improvement in muscle function being the most important end point in these trials, a lot of emphasis has been placed on setting up reliable, reproducible, and easy to perform functional tests to pre clinically assess muscle function, strength, condition, and coordination in the mdx
mouse model for DMD. Both invasive and noninvasive tests are available. Tests that do not exacerbate the disease can be used to determine the natural history of the disease and the effects of therapeutic interventions (e.g
. forelimb grip strength test, two different hanging tests using either a wire or a grid and rotarod running). Alternatively, forced treadmill running can be used to enhance disease progression and/or assess protective effects of therapeutic interventions on disease pathology. We here describe how to perform these most commonly used functional tests in a reliable and reproducible manner. Using these protocols based on standard operating procedures enables comparison of data between different laboratories.
Behavior, Issue 85, Duchenne muscular dystrophy, neuromuscular disorders, outcome measures, functional testing, mouse model, grip strength, hanging test wire, hanging test grid, rotarod running, treadmill running
Controlled Cervical Laceration Injury in Mice
Institutions: Norton Healthcare, Indiana University School of Medicine.
Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of ±0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.
Medicine, Issue 75, Neurobiology, Anatomy, Physiology, Neuroscience, Immunology, Infection, Surgery, Nervous System Diseases, Diagnosis, Therapeutics, Surgical Procedures, Operative, Investigative Techniques, spine, spinal cord injury, SCI, mouse, laceration, stabilization, axonal regeneration, injury, mice, animal model, surgical techniques
Anterior Cervical Discectomy and Fusion in the Ovine Model
Institutions: Monash University, Monash University.
Anterior cervical discectomy and fusion (ACDF) is the most common surgical operation for cervical radiculopathy and/or myelopathy in patients who have failed conservative treatment1,5
. Since the operation was first described by Cloward2
and Smith and Robinson6
in 1958, a variety refinements in technique, graft material and implants have been made3
. In particular, there is a need for safe osteoinductive agents that could benefit selected patients. The ovine model has been shown to have anatomical, biomechanical, bone density and radiological properties that are similar to the human counterpart, the most similar level being C3/44
. It is therefore an ideal model in which preclinical studies can be performed. In particular this methodology may be useful to researchers interested in evaluating different devices and biologics, including stem cells, for potential application in human spinal surgery.
Medicine, Issue 32, Anterior cervical discectomy, interbody fusion, spine fusion, stem cells, biologics, spine instrumentation, interbody cage
Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy
Institutions: Sainte-Justine University Hospital Research Center, Université de Montréal.
This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells' responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.
Medicine, Issue 80, Blood Cells, Lymphocytes, Spinal Diseases, Diagnostic Techniques and Procedures, Clinical Laboratory Techniques, Dielectric Spectroscopy, Musculoskeletal Diseases, Idiopathic scoliosis, classification, prognosis, G proteins, cellular dielectric spectroscopy, PBMCs
Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury
Institutions: Case Western Reserve University, Case Western Reserve University, Case Western Reserve University.
Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.
Cellular Biology, Issue 93, Intravital, spinal cord crush injury, chimera, microglia, macrophages, dorsal column crush, axonal dieback
An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time
Institutions: University of Louisville, University of Calgary.
Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo
living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo
spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo
spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g.,
calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo
); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.
Neuroscience, Issue 93, spinal cord injury, axon, myelin, two-photon excitation microscopy, Nile Red, axonal degeneration, axonal dieback, axonal retraction
Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents
Institutions: University of Alabama at Birmingham - UAB, University of Alabama at Birmingham - UAB, University of Alabama at Birmingham - UAB.
Although in vitro
screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1
and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2
. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2
. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13
and most commonly arise in the sciatic nerve4
. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.
Medicine, Issue 49, Orthotopic grafting, Schwann cell, sciatic nerve, MPNST, neurofibrosarcoma, neurofibromatosis, experimental therapeutics
Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro
using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro
. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo
. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo
tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro
and in vivo
and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells
Institutions: Medical College of Wisconsin.
Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in vitro
disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1
. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2–3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.
Medicine, Issue 95, Stem cell biology, Urine derived Cells (UCs), Induced Pluripotent Stem Cells (iPSCs), reprogramming, Sendai Virus (SeV), viral transduction, iPSC-derived Cardiomyocytes (iCMs), regenerative medicine, Muscular Dystrophy (MD), dystrophic cardiomyopathy.
Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90,
zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models
Institutions: University of Missouri, University of Missouri, University of Missouri.
This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e.,
high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.
Medicine, Issue 97, mouse, murine, rodent, swallowing, deglutition, dysphagia, videofluoroscopy, radiation, iohexol, barium, palatability, taste, translational, disease models
A Simple Critical-sized Femoral Defect Model in Mice
Institutions: Texas A&M Health Science Center, University of Texas Medical Branch, Texas A&M Health Science Center.
While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility.
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.
Medicine, Issue 97, Bone injury model, critical sized defect, mice, femur, tissue engineering, comparative medicine, medullary pin.
Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-γ-Dependent Cell Autonomous Immunity
Institutions: New York Medical College.
the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in naïve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.
Immunology, Issue 97, Toxoplasma, macrophages, innate immunity, intracellular pathogen, immune evasion, infectious disease, forward genetics, parasite
Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury
Institutions: Thomas Jefferson University Medical College.
Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1
and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3
. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4
. Transgenic mice 4,5
and rats 6
carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R)
have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7
. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1
. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11
. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A
mice and rats, as well as spinal cord injured rats and mice 11
Medicine, Issue 55, cell transplantation, engraftment, graft, spinal cord, stem cells, precursors, ALS, amyotrophic lateral sclerosis, motor neuron, SCI, spinal cord injury
Primary Culture of Human Vestibular Schwannomas
Institutions: University of Iowa Hospitals and Clinics.
Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor
gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.
Medicine, Issue 89, Primary Vestibular Schwannoma, Cranial Nerve Schwannoma, Primary Acoustic Neuroma, Cell Culture