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Long-term functional consequences and ongoing cerebral inflammation after subarachnoid hemorrhage in the rat.
PUBLISHED: 01-01-2014
Subarachnoid hemorrhage (SAH) represents a considerable health problem with an incidence of 6-7 per 100.000 individuals per year in Western society. We investigated the long-term consequences of SAH on behavior, neuroinflammation and gray- and white-matter damage using an endovascular puncture model in Wistar rats. Rats were divided into a mild or severe SAH group based on their acute neurological score at 24 h post-SAH. The degree of hemorrhage determined in post-mortem brains at 48 h strongly correlated with the acute neurological score. Severe SAH induced increased TNF-?, IL-1?, IL-10, MCP-1, MIP2, CINC-1 mRNA expression and cortical neutrophil influx at 48 h post-insult. Neuroinflammation after SAH was very long-lasting and still present at day 21 as determined by Iba-1 staining (microglia/macrophages) and GFAP (astrocytes). Long-term neuroinflammation was strongly associated with the degree of severity of SAH. Cerebral damage to gray- and white-matter was visualized by immunohistochemistry for MAP2 and MBP at 21 days after SAH. Severe SAH induced significant gray- and white-matter damage. MAP2 loss at day 21 correlated significantly with the acute neurological score determined at 24 h post-SAH. Sensorimotor behavior, determined by the adhesive removal task and von Frey test, was affected after severe SAH at day 21. In conclusion, we are the first to show that SAH induces ongoing cortical inflammation. Moreover, SAH induces mainly cortical long-term brain damage, which is associated with long-term sensorimotor damage.
Authors: Rahul V. Dudhani, Michele Kyle, Christina Dedeo, Margaret Riordan, Eric M. Deshaies.
Published: 01-17-2013
Objective: To characterize and establish a reproducible model that demonstrates delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) in rats, in order to identify the initiating events, pathophysiological changes and potential targets for treatment. Methods: Twenty-eight male Sprague-Dawley rats (250 - 300 g) were arbitrarily assigned to one of two groups - SAH or saline control. Rat subarachnoid hemorrhage in the SAH group (n=15) was induced by double injection of autologous blood, 48 hr apart, into the cisterna magna. Similarly, normal saline (n=13) was injected into the cisterna magna of the saline control group. Rats were sacrificed on day five after the second blood injection and the brains were preserved for histological analysis. The degree of vasospasm was measured using sections of the basilar artery, by measuring the internal luminal cross sectional area using NIH Image-J software. The significance was tested using Tukey/Kramer's statistical analysis. Results: After analysis of histological sections, basilar artery luminal cross sectional area were smaller in the SAH than in the saline group, consistent with cerebral vasospasm in the former group. In the SAH group, basilar artery internal area (.056 μm ± 3) were significantly smaller from vasospasm five days after the second blood injection (seven days after the initial blood injection), compared to the saline control group with internal area (.069 ± 3; p=0.004). There were no mortalities from cerebral vasospasm. Conclusion: The rat double SAH model induces a mild, survivable, basilar artery vasospasm that can be used to study the pathophysiological mechanisms of cerebral vasospasm in a small animal model. A low and acceptable mortality rate is a significant criterion to be satisfied for an ideal SAH animal model so that the mechanisms of vasospasm can be elucidated 7, 8. Further modifications of the model can be made to adjust for increased severity of vasospasm and neurological exams.
19 Related JoVE Articles!
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The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects
Authors: Lukas Andereggen, Volker Neuschmelting, Michael von Gunten, Hans Rudolf Widmer, Jukka Takala, Stephan M. Jakob, Javier Fandino, Serge Marbacher.
Institutions: University and Bern University Hospital (Inselspital), Kantonsspital Aarau, Boston Children's Hospital, Boston Children's Hospital, University and Bern University Hospital (Inselspital), University Hospital Cologne, Länggasse Bern.
Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH. This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.
Medicine, Issue 92, Subarachnoid hemorrhage, animal models, rabbit, extracorporeal blood shunt, early brain injury, delayed cerebral vasospasm, microsurgery.
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A Murine Model of Subarachnoid Hemorrhage
Authors: Kathrin Schüller, Dominik Bühler, Nikolaus Plesnila.
Institutions: University of Munich Medical Center.
In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.
Medicine, Issue 81, Nervous System Diseases, Subarachnoid hemorrhage (SAH), mouse model, filament perforation, intracranial pressure monitoring, blood distribution, surgical technique
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Controlled Cortical Impact Model for Traumatic Brain Injury
Authors: Jennifer Romine, Xiang Gao, Jinhui Chen.
Institutions: Indiana University School of Medicine.
Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.
Medicine, Issue 90, controlled cortical impact, traumatic brain injury, cortical contusion
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An Experimental Paradigm for the Prediction of Post-Operative Pain (PPOP)
Authors: Ruth Landau, John C. Kraft, Lisa Y. Flint, Brendan Carvalho, Philippe Richebé, Monica Cardoso, Patricia Lavand'homme, Michal Granot, David Yarnitsky, Alex Cahana.
Institutions: University of Washington School of Medicine.
Many women undergo cesarean delivery without problems, however some experience significant pain after cesarean section. Pain is associated with negative short-term and long-term effects on the mother. Prior to women undergoing surgery, can we predict who is at risk for developing significant postoperative pain and potentially prevent or minimize its negative consequences? These are the fundamental questions that a team from the University of Washington, Stanford University, the Catholic University in Brussels, Belgium, Santa Joana Women's Hospital in São Paulo, Brazil, and Rambam Medical Center in Israel is currently evaluating in an international research collaboration. The ultimate goal of this project is to provide optimal pain relief during and after cesarean section by offering individualized anesthetic care to women who appear to be more 'susceptible' to pain after surgery. A significant number of women experience moderate or severe acute post-partum pain after vaginal and cesarean deliveries. 1 Furthermore, 10-15% of women suffer chronic persistent pain after cesarean section. 2 With constant increase in cesarean rates in the US 3 and the already high rate in Brazil, this is bound to create a significant public health problem. When questioning women's fears and expectations from cesarean section, pain during and after it is their greatest concern. 4 Individual variability in severity of pain after vaginal or operative delivery is influenced by multiple factors including sensitivity to pain, psychological factors, age, and genetics. The unique birth experience leads to unpredictable requirements for analgesics, from 'none at all' to 'very high' doses of pain medication. Pain after cesarean section is an excellent model to study post-operative pain because it is performed on otherwise young and healthy women. Therefore, it is recommended to attenuate the pain during the acute phase because this may lead to chronic pain disorders. The impact of developing persistent pain is immense, since it may impair not only the ability of women to care for their child in the immediate postpartum period, but also their own well being for a long period of time. In a series of projects, an international research network is currently investigating the effect of pregnancy on pain modulation and ways to predict who will suffer acute severe pain and potentially chronic pain, by using simple pain tests and questionnaires in combination with genetic analysis. A relatively recent approach to investigate pain modulation is via the psychophysical measure of Diffuse Noxious Inhibitory Control (DNIC). This pain-modulating process is the neurophysiological basis for the well-known phenomenon of 'pain inhibits pain' from remote areas of the body. The DNIC paradigm has evolved recently into a clinical tool and simple test and has been shown to be a predictor of post-operative pain.5 Since pregnancy is associated with decreased pain sensitivity and/or enhanced processes of pain modulation, using tests that investigate pain modulation should provide a better understanding of the pathways involved with pregnancy-induced analgesia and may help predict pain outcomes during labor and delivery. For those women delivering by cesarean section, a DNIC test performed prior to surgery along with psychosocial questionnaires and genetic tests should enable one to identify women prone to suffer severe post-cesarean pain and persistent pain. These clinical tests should allow anesthesiologists to offer not only personalized medicine to women with the promise to improve well-being and satisfaction, but also a reduction in the overall cost of perioperative and long term care due to pain and suffering. On a larger scale, these tests that explore pain modulation may become bedside screening tests to predict the development of pain disorders following surgery.
JoVE Medicine, Issue 35, diffuse noxious inhibitory control, DNIC, temporal summation, TS, psychophysical testing, endogenous analgesia, pain modulation, pregnancy-induced analgesia, cesarean section, post-operative pain, prediction
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Investigating Intestinal Inflammation in DSS-induced Model of IBD
Authors: Janice J. Kim, Md. Sharif Shajib, Marcus M. Manocha, Waliul I. Khan.
Institutions: McMaster University .
Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2 There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6 The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8 In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.
Medicine, Issue 60, inflammation, myeloperoxidase (MPO), acute colonic damage, granulocyte, colon, dextran sulfate sodium (DSS), neutrophil
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Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion
Authors: Simone Beretta, Matteo Riva, Davide Carone, Elisa Cuccione, Giada Padovano, Virginia Rodriguez Menendez, Giovanni B. Pappadá, Alessandro Versace, Carlo Giussani, Erik P. Sganzerla, Carlo Ferrarese.
Institutions: University of Milano Bicocca.
The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.
Medicine, Issue 72, Neuroscience, Neurobiology, Biomedical Engineering, Anatomy, Physiology, Surgery, Brain Ischemia, Stroke, Hemodynamics, middle cerebral artery occlusion, cerebral hemodynamics, perfusion monitoring, Laser Doppler, intracranial collaterals, ischemic penumbra, rat, animal model
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Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging
Authors: Mikhail Paveliev, Mikhail Kislin, Dmitry Molotkov, Mikhail Yuryev, Heikki Rauvala, Leonard Khiroug.
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.
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Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage
Authors: Beilei Lei, Huaxin Sheng, Haichen Wang, Christopher D. Lascola, David S. Warner, Daniel T. Laskowitz, Michael L. James.
Institutions: Duke University, Duke University, Duke University, Duke University.
Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective treatment and failure of large clinical trials aimed at hemostasis and clot removal demonstrate the need for further mechanism-driven investigation of ICH. This research may be performed through the framework provided by preclinical models. Two murine models in popular use include intrastriatal (basal ganglia) injection of either autologous whole blood or clostridial collagenase. Since, each model represents distinctly different pathophysiological features related to ICH, use of a particular model may be selected based on what aspect of the disease is to be studied. For example, autologous blood injection most accurately represents the brain's response to the presence of intraparenchymal blood, and may most closely replicate lobar hemorrhage. Clostridial collagenase injection most accurately represents the small vessel rupture and hematoma evolution characteristic of deep hemorrhages. Thus, each model results in different hematoma formation, neuroinflammatory response, cerebral edema development, and neurobehavioral outcomes. Robustness of a purported therapeutic intervention can be best assessed using both models. In this protocol, induction of ICH using both models, immediate post-operative demonstration of injury, and early post-operative care techniques are demonstrated. Both models result in reproducible injuries, hematoma volumes, and neurobehavioral deficits. Because of the heterogeneity of human ICH, multiple preclinical models are needed to thoroughly explore pathophysiologic mechanisms and test potential therapeutic strategies.
Medicine, Issue 89, intracerebral hemorrhage, mouse, preclinical, autologous blood, collagenase, neuroscience, stroke, brain injury, basal ganglia
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Mouse Models of Periventricular Leukomalacia
Authors: Yan Shen, Jennifer M. Plane, Wenbin Deng.
Institutions: University of California, Davis.
We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.
JoVE Neuroscience, Issue 39, brain, mouse, white matter injury, oligodendrocyte, periventricular leukomalacia
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An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time
Authors: Starlyn L. M. Okada, Nicole S. Stivers, Peter K. Stys, David P. Stirling.
Institutions: University of Louisville, University of Calgary.
Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.
Neuroscience, Issue 93, spinal cord injury, axon, myelin, two-photon excitation microscopy, Nile Red, axonal degeneration, axonal dieback, axonal retraction
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Diffusion Tensor Magnetic Resonance Imaging in the Analysis of Neurodegenerative Diseases
Authors: Hans-Peter Müller, Jan Kassubek.
Institutions: University of Ulm.
Diffusion tensor imaging (DTI) techniques provide information on the microstructural processes of the cerebral white matter (WM) in vivo. The present applications are designed to investigate differences of WM involvement patterns in different brain diseases, especially neurodegenerative disorders, by use of different DTI analyses in comparison with matched controls. DTI data analysis is performed in a variate fashion, i.e. voxelwise comparison of regional diffusion direction-based metrics such as fractional anisotropy (FA), together with fiber tracking (FT) accompanied by tractwise fractional anisotropy statistics (TFAS) at the group level in order to identify differences in FA along WM structures, aiming at the definition of regional patterns of WM alterations at the group level. Transformation into a stereotaxic standard space is a prerequisite for group studies and requires thorough data processing to preserve directional inter-dependencies. The present applications show optimized technical approaches for this preservation of quantitative and directional information during spatial normalization in data analyses at the group level. On this basis, FT techniques can be applied to group averaged data in order to quantify metrics information as defined by FT. Additionally, application of DTI methods, i.e. differences in FA-maps after stereotaxic alignment, in a longitudinal analysis at an individual subject basis reveal information about the progression of neurological disorders. Further quality improvement of DTI based results can be obtained during preprocessing by application of a controlled elimination of gradient directions with high noise levels. In summary, DTI is used to define a distinct WM pathoanatomy of different brain diseases by the combination of whole brain-based and tract-based DTI analysis.
Medicine, Issue 77, Neuroscience, Neurobiology, Molecular Biology, Biomedical Engineering, Anatomy, Physiology, Neurodegenerative Diseases, nuclear magnetic resonance, NMR, MR, MRI, diffusion tensor imaging, fiber tracking, group level comparison, neurodegenerative diseases, brain, imaging, clinical techniques
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Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Authors: Aya D. Pusic, Yelena Y. Grinberg, Heidi M. Mitchell, Richard P. Kraig.
Institutions: The University of Chicago Medical Center, The University of Chicago Medical Center.
Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.
Neuroscience, Issue 52, innate immunity, hormesis, microglia, T-cells, hippocampus, slice culture, gene expression, laser dissection microscopy, real-time qPCR, interferon-gamma
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Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Authors: Robert S. McNeill, Ralf S. Schmid, Ryan E. Bash, Mark Vitucci, Kristen K. White, Andrea M. Werneke, Brian H. Constance, Byron Huff, C. Ryan Miller.
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
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Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery
Authors: Gemma Llovera, Stefan Roth, Nikolaus Plesnila, Roland Veltkamp, Arthur Liesz.
Institutions: University Hospital Munich, Munich Cluster for Systems Neurology (SyNergy), University Heidelberg, Charing Cross Hospital.
Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called “coagulation model”. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the “coagulation model” and report histological and functional analysis tools.
Medicine, Issue 89, stroke, brain ischemia, animal model, middle cerebral artery, electrocoagulation
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Permanent Cerebral Vessel Occlusion via Double Ligature and Transection
Authors: Melissa F. Davis, Christopher Lay, Ron D. Frostig.
Institutions: University of California, Irvine, University of California, Irvine, University of California, Irvine, University of California, Irvine.
Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA. Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.
Medicine, Issue 77, Biomedical Engineering, Anatomy, Physiology, Neurobiology, Neuroscience, Behavior, Surgery, Therapeutics, Surgical Procedures, Operative, Investigative Techniques, Life Sciences (General), Behavioral Sciences, Animal models, Stroke, ischemia, imaging, middle cerebral artery, vessel occlusion, rodent model, surgical techniques, animal model
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Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Authors: Yves Molino, Françoise Jabès, Emmanuelle Lacassagne, Nicolas Gaudin, Michel Khrestchatisky.
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),
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Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)
Authors: Thomas Lenz, Peter Poot, Olivia Gräbner, Mirko Glinski, Elmar Weinhold, Mathias Dreger, Hubert Köster.
Institutions: caprotec bioanalytics GmbH, RWTH Aachen University.
There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules). S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A). SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5α of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.
Biochemistry, Issue 46, Capture Compound, photo-crosslink, small molecule-protein interaction, methyltransferase, S-adenosyl-l-homocysteine, SAH, S-adenosyl-l-methionine, SAM, functional proteomics, LC-MS/MS
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Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining
Authors: Estelle Rousselet, Jasna Kriz, Nabil G. Seidah.
Institutions: Clinical Research Institute of Montreal, Laval University.
Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3. Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.
Medicine, Issue 69, Neuroscience, Biochemistry, Anatomy, Physiology, transient ischemic stroke, middle cerebral artery occlusion, intraluminal model, neuroscore, cresyl violet staining, mice, imaging
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The Ladder Rung Walking Task: A Scoring System and its Practical Application.
Authors: Gerlinde A. Metz, Ian Q. Whishaw.
Institutions: University of Lethbridge.
Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb’s misplacement.
Neuroscience, Issue 28, rat, animal model of walking, skilled movement, ladder test, rung test, neuroscience
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