Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.
25 Related JoVE Articles!
Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging
Institutions: University of Helsinki.
Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo
. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.
Medicine, Issue 86, Trauma, Nervous System, animal models, Brain trauma, in vivo multiphoton microscopy, dendrite, astrocyte, microglia, second harmonic generation.
The 5-Choice Serial Reaction Time Task: A Task of Attention and Impulse Control for Rodents
Institutions: Oberlin College.
This protocol describes the 5-choice serial reaction time task, which is an operant based task used to study attention and impulse control in rodents. Test day challenges, modifications to the standard task, can be used to systematically tax the neural systems controlling either attention or impulse control. Importantly, these challenges have consistent effects on behavior across laboratories in intact animals and can reveal either enhancements or deficits in cognitive function that are not apparent when rats are only tested on the standard task. The variety of behavioral measures that are collected can be used to determine if other factors (i.e
., sedation, motivation deficits, locomotor impairments) are contributing to changes in performance. The versatility of the 5CSRTT is further enhanced because it is amenable to combination with pharmacological, molecular, and genetic techniques.
Neuroscience, Issue 90, attention, impulse control, neuroscience, cognition, rodent
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)
Remote Limb Ischemic Preconditioning: A Neuroprotective Technique in Rodents
Institutions: University of Sydney.
Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb – specifically the hind limb of a rat – ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.
Medicine, Issue 100, remote ischemic preconditioning, ischemic conditioning, ischemic tolerance, light injury, neuroprotection, mediated neuroprotection, stroke, retina, electroretinogram, rat
Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina
Institutions: University Eye Hospital Tübingen.
Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing.
Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N2
) or glutamate stress (250 µm glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min.
For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p <0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p = 0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p <0.01, decrease of 25.5% at the end of the washout, p = 0.03).
For 250 µm glutamate, an initial 7.8% reduction of a-wave amplitudes (p >0.05) followed by a reduction of 1.9% (p >0.05). A reduction of 83.7% of b-wave amplitudes (p <0.01) was noted; after a washout of 75 min the reduction was 2.3% (p = 0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.
Medicine, Issue 97, Glutamate, Hypoxia, retinal toxicity, electroretinogram, intraocular toxicity, superfused retina
Laser-Induced Chronic Ocular Hypertension Model on SD Rats
Institutions: The University of Hong Kong - HKU.
Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.
Neuroscience, Issue 10, glaucoma, ocular hypertension, rat
The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats
Institutions: Utrecht University, Brain Center Rudolf Magnus.
This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g.,
mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal’s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the ‘protected’ area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the ‘unprotected’ area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals’ adaptive capacities.
Due to testing order effects in a behavioral test battery, naïve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.
Behavior, Issue 98, Anxiety, behavior, cognition, exploration, locomotion, mice, modified hole board, rats, social interaction.
Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning
Institutions: University of Sydney.
The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the ‘flash’ ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.
Neuroscience, Issue 100, remote ischemic preconditioning, ischemic tolerance, ischemic preconditioning, neuroprotection, retinal degeneration, light damage, photoreceptors, retina, electroretinogram, rat, mouse
Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice
Institutions: University of Texas Southwestern Medical Center, Washington University School of Medicine.
Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2
). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2
room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.
Medicine, Issue 99, Hypoxia, preconditioning, transient middle cerebral artery occlusion, stroke, neuroprotection, blood-brain barrier disruption
In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma
Institutions: University of Utah, University of Utah.
Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo
imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+
reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo
images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.
Medicine, Issue 99, Neuroscience, microglia, neurodegeneration, glaucoma, retina, optic nerve head, confocal scanning laser ophthalmoscopy, live image analysis, segmentation by thresholding, cell morphometry CX3CR1, DBA/2J
Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus
Institutions: Columbia University Medical Center, Columbia University Medical Center, Columbia University Medical Center.
Alzheimer’s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer’s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer’s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.
Neuroscience, Issue 100, Amyloid-beta, brain, mouse, infusion, surgery, neuroscience
A Coupled Experiment-finite Element Modeling Methodology for Assessing High Strain Rate Mechanical Response of Soft Biomaterials
Institutions: Mississippi State University, Mississippi State University.
This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g.
brain, liver, tendon, fat, etc.
) when exposed to high strain rates. This study utilized a Split-Hopkinson Pressure Bar (SHPB) to generate strain rates of 100-1,500 sec-1
. The SHPB employed a striker bar consisting of a viscoelastic material (polycarbonate). A sample of the biomaterial was obtained shortly postmortem and prepared for SHPB testing. The specimen was interposed between the incident and transmitted bars, and the pneumatic components of the SHPB were activated to drive the striker bar toward the incident bar. The resulting impact generated a compressive stress wave (i.e.
incident wave) that traveled through the incident bar. When the compressive stress wave reached the end of the incident bar, a portion continued forward through the sample and transmitted bar (i.e.
transmitted wave) while another portion reversed through the incident bar as a tensile wave (i.e.
reflected wave). These waves were measured using strain gages mounted on the incident and transmitted bars. The true stress-strain behavior of the sample was determined from equations based on wave propagation and dynamic force equilibrium. The experimental stress-strain response was three dimensional in nature because the specimen bulged. As such, the hydrostatic stress (first invariant) was used to generate the stress-strain response. In order to extract the uniaxial (one-dimensional) mechanical response of the tissue, an iterative coupled optimization was performed using experimental results and Finite Element Analysis (FEA), which contained an Internal State Variable (ISV) material model used for the tissue. The ISV material model used in the FE simulations of the experimental setup was iteratively calibrated (i.e.
optimized) to the experimental data such that the experiment and FEA strain gage values and first invariant of stresses were in good agreement.
Bioengineering, Issue 99, Split-Hopkinson Pressure Bar, High Strain Rate, Finite Element Modeling, Soft Biomaterials, Dynamic Experiments, Internal State Variable Modeling, Brain, Liver, Tendon, Fat
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
Retrograde Labeling of Retinal Ganglion Cells by Application of Fluoro-Gold on the Surface of Superior Colliculus
Institutions: The University of Hong Kong - HKU.
Retinal ganglion cell (RGC) counting is essential to evaluate retinal degeneration especially in glaucoma. Reliable RGC labeling is fundamental for evaluating the effects of any treatment. In rat, about 98% of RGCs is known to project to the contralateral superior colliculus (SC) (Forrester and Peters, 1967). Applying fluoro-gold (FG) on the surface of SC can label almost all the RGCs, so that we can focus on this most vulnerable retinal neuron in glaucoma. FG is taken up by the axon terminals of retinal ganglion cells and bilaterally transported retrogradely to its somas in the retina. Compare with retrograde labeling of RGC by putting FG at stump of transected optic nerve for 2 days, the interference of RGC survival is minimized. Compare with cresyl violet staining that stains RGCs, amacrine cells and endothelium of the blood vessel in the retinal ganglion cell layer, this labeling method is more specific to the RGC. This video describes the method of retrograde labeling of RGC by applying FG on the surface of SC. The surgical procedures include drilling the skull; aspirating the cortex to expose the SC and applying gelatin sponge over entire dorsal surface of SC are shown. Useful tips for avoiding massive intracranial bleeding and aspiration of the SC have been given.
Neuroscience, Issue 16, Retrograde labeling, retinal ganglion cells, ophthalmology research, superior colliculus, experimental glaucoma
A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila
Institutions: King's College London.
eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila
eye to address fundamentally important biological questions.
Cellular Biology, Issue 34, fluorescence microscopy, Drosophila, eye, RFP, dissection, imaginal disc
Isolation of Retinal Stem Cells from the Mouse Eye
Institutions: University of Toronto.
The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3
. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4
; however the cells are still capable of forming the different cell types found within the neural retina1-3
. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5
, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo
and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8
, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12
. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo
, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.
Cellular Biology, Issue 43, Stem Cells, Eye, Ciliary Epithelium, Tissue Culture, Mouse
Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium
Institutions: Greehey Children's Cancer Research Institute and Department of Cellular and Structural Biology.
The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo
assay of homology-directed DNA repair, the pun
assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun
assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.
Neuroscience, Issue 48, mouse, dissection, eye, retinal pigment epithelium, flat mount, whole mount, RPE
An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival
Institutions: NIH, The Second Hospital of Harbin Medical University.
Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.
Neuroscience, Issue 50, optic nerve crush injury, retinal ganglion cell, glaucoma, optic neuropathy, retrograde labeling
Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study
Institutions: University of Nebraska-Lincoln.
A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). We demonstrate in this video article how blast TBI-relevant impulsive pressurization is applied to the neuronal cells in vitro. This is achieved by using well-controlled pressure pulse created by a specialized Kolsky bar device, with complete pressure history within the cell pressurization chamber recorded. Pressurized neuronal cells are inspected immediately after pressurization, or further incubated to examine the long-term effects of impulsive pressurization on neurite/axonal outgrowth, neuronal gene expression, apoptosis, etc. We observed that impulsive pressurization at about 2 MPa induces distinct neurite loss relative to unpressurized cells. Our technique provides a novel method to investigate the molecular/cellular mechanisms of blast TBI, via impulsive pressurization of brain cells at well-controlled pressure magnitude and duration.
Bioengineering, Issue 56, Neuroscience, Traumatic Brain Injury, Neuronal Cells, Neurons, Impulsive Pressurization, Blast-TBI
Institutions: University of Utah.
A limitation of traditional full-field electroretinograms (ERG) for the diagnosis of retinopathy is lack of sensitivity. Generally, ERG results are normal unless more than approximately 20% of the retina is affected. In practical terms, a patient might be legally blind as a result of macular degeneration or other scotomas and still appear normal, according to traditional full field ERG. An important development in ERGs is the multifocal ERG (mfERG). Erich Sutter adapted the mathematical sequences called binary m-sequences enabling the isolation from a single electrical signal an electroretinogram representing less than each square millimeter of retina in response to a visual stimulus1
Results that are generated by mfERG appear similar to those generated by flash ERG. In contrast to flash ERG, which best generates data appropriate for whole-eye disorders. The basic mfERG result is based on the calculated mathematical average of an approximation of the positive deflection component of traditional ERG response, known as the b-wave1
. Multifocal ERG programs measure electrical activity from more than a hundred retinal areas per eye, in a few minutes. The enhanced spatial resolution enables scotomas and retinal dysfunction to be mapped and quantified.
In the protocol below, we describe the recording of mfERGs using a bipolar speculum contact lens.
Components of mfERG systems vary between manufacturers. For the presentation of visible stimulus, some suitable CRT monitors are available but most systems have adopted the use of flat-panel liquid crystal displays (LCD). The visual stimuli depicted here, were produced by a LCD microdisplay subtending 35 - 40 degrees horizontally and 30 - 35 degrees vertically of visual field, and calibrated to produce multifocal flash intensities of 2.7 cd s m-2
. Amplification was 50K. Lower and upper bandpass limits were 10 and 300 Hz. The software packages used were VERIS versions 5 and 6.
Medicine, Issue 58, Multifocal electroretinogram, mfERG, electroretinogram, ERG
Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye
Institutions: Columbia University , Columbia University , University of Iowa , University of Iowa .
The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1
Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2
These include RPE65
gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4
Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8
In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.
Stem Cell Biology, Issue 69, Medicine, Ophthalmology, Anatomy, Physiology, Cellular Biology, Genetics, mouse, subretinal injection, iPS cells, stem cells, retina, eye, gene therapy
A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects
Institutions: Northwestern University, Northwestern University.
Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.
Medicine, Issue 78, Biomedical Engineering, Neurobiology, Anatomy, Physiology, Neuroscience, Cellular Biology, Molecular Biology, Ophthalmology, Retinal Neurons, Retinal Neurons, Retinal Ganglion Cells, Neurodegenerative Diseases, Ocular Hypertension, Retinal Degeneration, Vision Tests, Visual Acuity, Eye Diseases, Retinal Ganglion Cell (RGC), Ocular Hypertension, Laser Photocoagulation, Intraocular pressure (IOP), Tonometer; Visual Acuity, Contrast Sensitivity, Optomotor, animal model
Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells
Institutions: KU Leuven.
Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+
-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3
that initiate the propagation of the Ca2+
-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+
-wave propagation are provided by gap junction channels through the direct transfer of IP3
and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+
-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+
-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+
-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.
Cellular Biology, Issue 77, Molecular Biology, Medicine, Biomedical Engineering, Biophysics, Immunology, Ophthalmology, Gap Junctions, Connexins, Connexin 43, Calcium Signaling, Ca2+, Cell Communication, Paracrine Communication, Intercellular communication, calcium wave propagation, gap junctions, hemichannels, endothelial cells, cell signaling, cell, isolation, cell culture
Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease
Institutions: SRI International, University of California-Santa Cruz.
Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer's disease, but its effects in Parkinson's disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone. To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e.
5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder. Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD. The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates. This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.
Medicine, Issue 83, MPTP, dopamine, Iba1, TH, GFAP, lipoxygenase, transgenic, gene-environment interactions, mouse, Parkinson's disease, neurodegeneration, neuroinflammation
Establishment and Characterization of UTI and CAUTI in a Mouse Model
Institutions: Washington University School of Medicine.
Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli
(UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis
. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis
bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.
Medicine, Issue 100, Escherichia coli, UPEC, Enterococcus faecalis, uropathogenic, catheter, urinary tract infection, IBC, chronic cystitis