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.
26 Related JoVE Articles!
Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons
Institutions: Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine.
It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2
. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3
. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4
. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro
. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8
. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro
model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9
Neuroscience, Issue 66, Physiology, Developmental Biology, cell culture, axon regeneration, axon growth, dorsal root ganglion, spinal cord injury
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
Revealing Neural Circuit Topography in Multi-Color
Institutions: Yeshiva University.
Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably label the global patterning of multiple topographic projections. The cerebellum is an ideal model to study the orderly arrangement of neural circuits. For example, the compartmental organization of spinocerebellar mossy fibers has proven to be an indispensable system for studying mossy fiber patterning. We recently showed that wheat germ agglutinin (WGA) conjugated to Alexa 555 and 488 can be used for tracing spinocerebellar mossy fiber projections in developing and adult mice (Reeber et al. 2011). We found three major properties that make the WGA-Alexa tracers desirable tools for labeling neural projections. First, Alexa fluorophores are intense and their brightness allows for wholemount imaging directly after tracing. Second, WGA-Alexa tracers label the entire trajectory of developing and adult neural projections. Third, WGA-Alexa tracers are rapidly transported in both retrograde and anterograde directions. Here, we describe in detail how to prepare the tracers and other required tools, how to perform the surgery for spinocerebellar tracing and how best to image traced projections in three dimensions. In summary, we provide a step-by-step tracing protocol that will be useful for deciphering the organization and connectivity of functional maps not only in the cerebellum but also in the cortex, brainstem, and spinal cord.
Neuroscience, Issue 57, neuronal projections, topography, circuits, connectivity, fluorescent tracers, mice
A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt
Institutions: University of Dayton, University of Dayton.
Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1
. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3
. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro
cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1
). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo
process (Figure 2
. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.
Cellular Biology, Issue 52, IPE cells, lens, regeneration, newt
Mechanical Testing of Mouse Carotid Arteries: from Newborn to Adult
Institutions: Saint Louis University.
The large conducting arteries in vertebrates are composed of a specialized extracellular matrix designed to provide pulse dampening and reduce the work performed by the heart. The mix of matrix proteins determines the passive mechanical properties of the arterial wall1
. When the matrix proteins are altered in development, aging, disease or injury, the arterial wall remodels, changing the mechanical properties and leading to subsequent cardiac adaptation2
. In normal development, the remodeling leads to a functional cardiac and cardiovascular system optimized for the needs of the adult organism. In disease, the remodeling often leads to a negative feedback cycle that can cause cardiac failure and death. By quantifying passive arterial mechanical properties in development and disease, we can begin to understand the normal remodeling process to recreate it in tissue engineering and the pathological remodeling process to test disease treatments.
Mice are useful models for studying passive arterial mechanics in development and disease. They have a relatively short lifespan (mature adults by 3 months and aged adults by 2 years), so developmental3
and aging studies4
can be carried out over a limited time course. The advances in mouse genetics provide numerous genotypes and phenotypes to study changes in arterial mechanics with disease progression5
and disease treatment6
. Mice can also be manipulated experimentally to study the effects of changes in hemodynamic parameters on the arterial remodeling process7
. One drawback of the mouse model, especially for examining young ages, is the size of the arteries.
We describe a method for passive mechanical testing of carotid arteries from mice aged 3 days to adult (approximately 90 days). We adapt a commercial myograph system to mount the arteries and perform multiple pressure or axial stretch protocols on each specimen. We discuss suitable protocols for each age, the necessary measurements and provide example data. We also include data analysis strategies for rigorous mechanical characterization of the arteries.
Bioengineering, Issue 60, blood vessel, artery, mechanics, pressure, diameter, postnatal development
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),
In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint
Institutions: University of California San Francisco, University of California San Francisco, Xradia Inc..
This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ
loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e.
from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo
conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.
Bioengineering, Issue 85, biomechanics, bone-periodontal ligament-tooth complex, concentric loads, eccentric loads, contrast agent
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
Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.
Institutions: Harvard Medical School, Massachusetts Eye and Ear Infirmary, Emerson College, Harvard.
In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro
culture of the organ explant 1,2,9
. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro
in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.
Neuroscience, Issue 36, hearing, mice, cochlea, organ of Corti, organotypic, culture, hair cell, stem cell, gene expression, in vitro
Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications
Institutions: Southern Illinois University School of Medicine, Southern Illinois University School of Medicine, Southern Illinois University Carbondale, University of Illinois at Springfield.
Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 ºC or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.
Bioengineering, Issue 86, Anterior Cruciate Ligament, Tissue Engineering, hACL derived cells, PLAGA, in vitro expansion, ACL partial tears
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
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
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
A Contusion Model of Severe Spinal Cord Injury in Rats
Institutions: Medical University of South Carolina, Clemson University, Clemson-MUSC Bioengineering Joint Program.
The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.
Biomedical Engineering, Issue 78, Medicine, Neurobiology, Neuroscience, Anatomy, Physiology, Surgery, Cerebrovascular Trauma, Spinal Cord Injuries, spinal cord injury model, contusion spinal cord injury, spinal cord contusion, translational spinal cord injury model, animal model
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
Designing Silk-silk Protein Alloy Materials for Biomedical Applications
Institutions: Rowan University, Rowan University, Cooper Medical School of Rowan University, Rowan University.
Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi
) and domestic mulberry silk (Bombyx mori
) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.
Bioengineering, Issue 90, protein alloys, biomaterials, biomedical, silk blends, computational simulation, implantable electronic devices
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)
Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises
Institutions: University of Kentucky, University of Kentucky, University of Oregon.
The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.
Neuroscience, Issue 80, Crustacean, joint, Muscle, sensory, teaching, educational, neuroscience
High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology
Institutions: University of Pittsburgh School of Medicine, University of Pittsburgh School of Medicine.
We describe an in vitro
method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo
experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e.
smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo
. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro
smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.
Medicine, Issue 90, Krebs, species differences, in vitro, smooth muscle contractility, neural stimulation
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
Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound
Institutions: Johns Hopkins University, Johns Hopkins University, Johns Hopkins University, Macquarie University.
We present a protocol for measuring in vivo
aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo
approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.
Medicine, Issue 94, Aortic stiffness, ultrasound, in vivo, aortic compliance, elastic modulus, mouse model, cardiovascular disease
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
In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice
Institutions: Jena University Hospital, Fritz Lipmann Institute, Jena, Jena University Hospital.
The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo
visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 µm)3
can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2
leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+
transport completely arrests at the lesion site. Conversely, active Mn2+
transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+
transport kinetics along the visual path in a transgenic mouse model (NF-κB p50KO
) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+
transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-κB mutations.
In summary, MEMRI conveniently bridges in vivo
assays and post mortem
histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.
Neuroscience, Issue 89, manganese-enhanced MRI, mouse retino-tectal projection, visual system, neurodegeneration, optic nerve injury, NF-κB
In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos
Institutions: University of California, San Francisco - UCSF.
In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.
Neuroscience, Issue 6, Protocol, Stem Cells, Cerebral Cortex, Brain Development, Electroporation, Intra Uterine Injections, transfection
Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties
Institutions: Weill Cornell Medical College, Weill Cornell Medical College of Cornell University.
Membrane protein function is regulated by the cell membrane lipid composition. This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function. The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid. Hence the gramicidin channel current is a reporter of altered properties of the bilayer due to certain compounds.
Cellular Biology, Issue 21, Springer Protocols, Membrane Biophysics, Gramicidin Channels, Artificial Bilayers, Bilayer Elastic Properties,