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.
22 Related JoVE Articles!
Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat
Institutions: University of Wisconsin-Madison.
Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1
. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3
. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.
Neuroscience, Issue 48, Stroke, cerebral ischemia, middle cerebral artery occlusion, intraluminal filament, rat, magnetic resonance imaging, surgery, neuroscience, brain
The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse
Institutions: University of Rochester, University of Alabama at Birmingham, University of Rochester.
Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6
. This method was originally described for rats by Tamura et al. in 1981 7
. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13
. Early use of this technique led to the definition and description of “infarct core” and “penumbra” 14-16
, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17
. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21
and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.
Medicine, Issue 53, brain, stroke, mouse, middle cerebral artery ligation
Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining
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
Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model
Institutions: Center for Stroke Research Berlin, Charité Universitätsmedizin.
Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.
Medicine, Issue 47, Stroke, middle cerebral artery occlusion, MCAo, animal model, mouse, techniques
Mouse Models of Periventricular Leukomalacia
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
Compensatory Limb Use and Behavioral Assessment of Motor Skill Learning Following Sensorimotor Cortex Injury in a Mouse Model of Ischemic Stroke
Institutions: Illinois Wesleyan University, University of Victoria.
Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. The present protocol describes a skilled motor task for use in mouse models of stroke. The Pasta Matrix Reaching Task functions as a versatile and sensitive behavioral assay that permits experimenters to collect accurate outcome data and manipulate limb use to mimic human clinical phenomena including compensatory strategies (i.e.
, learned non-use) and focused rehabilitative training. When combined with neuroanatomical tools, this task also permits researchers to explore the mechanisms that support behavioral recovery of function (or lack thereof) following stroke. The task is both simple and affordable to set up and conduct, offering a variety of training and testing options for numerous research questions concerning functional outcome following injury. Though the task has been applied to mouse models of stroke, it may also be beneficial in studies of functional outcome in other upper extremity injury models.
Behavior, Issue 89, Upper extremity impairment, Murine model, Rehabilitation, Reaching, Non-paretic limb training, Good limb training, Less-affected limb training, Learned non-use, Pasta matrix reaching task
Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia
Institutions: Charité - Universitätsmedizin Berlin, Germany.
There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.
Medicine, Issue 75, Neurobiology, Anatomy, Physiology, Neuroscience, Immunology, Surgery, stroke, cerebral ischemia, ischemic preconditioning, ischemic tolerance, IT, ischemic stroke, middle cerebral artery occlusion, MCAO, bilateral common carotid artery occlusion, BCCAO, brain, ischemia, occlusion, reperfusion, mice, animal model, surgical techniques
2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia
Institutions: Wayne State University School of Medicine, Wayne State University School of Medicine, Wayne State University School of Medicine.
Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1
. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al
. (1984) 2
, as first presented in its current form in Sanderson, et al.
, which produces reproducible injury to selectively vulnerable brain regions 3-6
. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.
Medicine, Issue 76, Biomedical Engineering, Neurobiology, Neuroscience, Immunology, Anatomy, Physiology, Cardiology, Brain Ischemia, ischemia, reperfusion, cardiac arrest, resuscitation, 2VOH, brain injury model, CA1 hippocampal neurons, brain, neuron, blood vessel, occlusion, hypotension, animal model
Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury
Institutions: IRCCS - Istituto di Ricerche Farmacologiche Mario Negri.
After brain stroke microglia/macrophages (M/M) undergo rapid activation with dramatic morphological and phenotypic changes that include expression of novel surface antigens and production of mediators that build up and maintain the inflammatory response. Emerging evidence indicates that M/M are highly plastic cells that can assume classic pro-inflammatory (M1) or alternative anti-inflammatory (M2) activation after acute brain injury. However a complete characterization of M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is still missing.
Immunofluorescence protocols specifically staining relevant markers of M/M activation can be performed in the ischemic brain. Here we present immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate the pattern of localization and co-expression of M/M phenotype markers such as CD11b, CD68, Ym1, in mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery (pMCAO). Two-dimensional analysis of the stained area reveals that each marker is associated to a defined M/M morphology and has a given localization in the ischemic lesion. Patterns of M/M phenotype marker co-expression can be assessed by three-dimensional confocal imaging in the ischemic area. Images can be acquired over a defined volume (10 μm z-axis and a 0.23 μm step size, corresponding to a 180 x 135 x 10 μm volume) with a sequential scanning mode to minimize bleed-through effects and avoid wavelength overlapping. Images are then processed to obtain three-dimensional renderings by means of Imaris software. Solid view of three dimensional renderings allows the definition of marker expression in clusters of cells. We show that M/M have the ability to differentiate towards a multitude of phenotypes, depending on the location in the lesion site and time after injury.
Neurobiology, Issue 79, Neuroscience, Molecular Biology, Cellular Biology, Medicine, Biomedical Engineering, Anatomy, Physiology, Central Nervous System Diseases, Neurodegenerative Diseases, biology (general), immunology, life sciences, animal models, Inflammation, stroke, alternative activation, brain injury, brain, imaging, confocal microscopy, three-dimensional imaging, clinical techniques, mouse, animal model
The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia
Institutions: Stanford University School of Medicine.
Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1
and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2
and to test potential therapeutic interventions 3,4
. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5
, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6
. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.
Neuroscience, Issue 21, Hypoxic-ischemic encephalopathy (HIE), 2 3 5-triphenyltetrazolium chloride (TTC), brain infarct
Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier
Institutions: Monash University.
The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro
BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.
Bioengineering, Issue 81, Blood-brain barrier, model, cell culture, astrocytes, brain endothelial cells, insert, membranes
Mouse Model of Middle Cerebral Artery Occlusion
Institutions: Ernest Gallo Clinic and Research Center, University of California, San Francisco, Kent State University.
Stroke is the most common fatal neurological disease in the United States 1
. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2
. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3
, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4
. The technique can be used to model permanent or transient occlusion 5
. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6
. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8
. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9
. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.
Medicine, Issue 48, Neurology, Stroke, mice, ischemia
Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats
Institutions: Louisiana State University Health Science Center, Shreveport.
Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1
in 1992 and further characterized by Zhang et al.
. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.
Medicine, Issue 91, ischemic stroke, model, embolus, middle cerebral artery occlusion, thrombolytic therapy
Permanent Cerebral Vessel Occlusion via Double Ligature and Transection
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
Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery
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
Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly
Institutions: Sunnybrook Health Sciences Centre, University of Toronto.
Obtaining in vivo
human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g.
Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2
. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.
Medicine, Issue 86, Brain, Vascular Diseases, Magnetic Resonance Imaging (MRI), Neuroimaging, Alzheimer Disease, Aging, Neuroanatomy, brain extraction, ventricles, white matter hyperintensities, cerebrovascular disease, Alzheimer disease
Breathing-controlled Electrical Stimulation (BreEStim) for Management of Neuropathic Pain and Spasticity
Institutions: University of Texas Health Science Center at Houston , TIRR Memorial Hermann Hospital, TIRR Memorial Hermann Hospital.
Electrical stimulation (EStim) refers to the application of electrical current to muscles or nerves in order to achieve functional and therapeutic goals. It has been extensively used in various clinical settings. Based upon recent discoveries related to the systemic effects of voluntary breathing and intrinsic physiological interactions among systems during voluntary breathing, a new EStim protocol, Breathing-controlled Electrical Stimulation (BreEStim), has been developed to augment the effects of electrical stimulation. In BreEStim, a single-pulse electrical stimulus is triggered and delivered to the target area when the airflow rate of an isolated voluntary inspiration reaches the threshold. BreEStim integrates intrinsic physiological interactions that are activated during voluntary breathing and has demonstrated excellent clinical efficacy. Two representative applications of BreEStim are reported with detailed protocols: management of post-stroke finger flexor spasticity and neuropathic pain in spinal cord injury.
Medicine, Issue 71, Neuroscience, Neurobiology, Anatomy, Physiology, Behavior, electrical stimulation, BreEStim, electrode, voluntary breathing, respiration, inspiration, pain, neuropathic pain, pain management, spasticity, stroke, spinal cord injury, brain, central nervous system, CNS, clinical, electromyogram, neuromuscular electrical stimulation
The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism
Institutions: University of Montréal, McGill University, University of Minnesota.
Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33
. To help improve this understanding, proton magnetic resonance spectroscopy (1
H-MRS) can be used as it allows the in vivo
quantification of brain metabolites such as γ-aminobutyric acid (GABA) and glutamate in a region-specific manner 41
. In fact, a recent study demonstrated that 1
H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34
. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1
H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31
. Methodological factors to consider and possible modifications to the protocol are also discussed.
Neuroscience, Issue 93, proton magnetic resonance spectroscopy, transcranial direct current stimulation, primary motor cortex, GABA, glutamate, stroke
Identification of Disease-related Spatial Covariance Patterns using Neuroimaging Data
Institutions: The Feinstein Institute for Medical Research.
The scaled subprofile model (SSM)1-4
is a multivariate PCA-based algorithm that identifies major sources of variation in patient and control group brain image data while rejecting lesser components (Figure 1
). Applied directly to voxel-by-voxel covariance data of steady-state multimodality images, an entire group image set can be reduced to a few significant linearly independent covariance patterns and corresponding subject scores. Each pattern, termed a group invariant subprofile (GIS), is an orthogonal principal component that represents a spatially distributed network of functionally interrelated brain regions. Large global mean scalar effects that can obscure smaller network-specific contributions are removed by the inherent logarithmic conversion and mean centering of the data2,5,6
. Subjects express each of these patterns to a variable degree represented by a simple scalar score that can correlate with independent clinical or psychometric descriptors7,8
. Using logistic regression analysis of subject scores (i.e.
pattern expression values), linear coefficients can be derived to combine multiple principal components into single disease-related spatial covariance patterns, i.e.
composite networks with improved discrimination of patients from healthy control subjects5,6
. Cross-validation within the derivation set can be performed using bootstrap resampling techniques9
. Forward validation is easily confirmed by direct score evaluation of the derived patterns in prospective datasets10
. Once validated, disease-related patterns can be used to score individual patients with respect to a fixed reference sample, often the set of healthy subjects that was used (with the disease group) in the original pattern derivation11
. These standardized values can in turn be used to assist in differential diagnosis12,13
and to assess disease progression and treatment effects at the network level7,14-16
. We present an example of the application of this methodology to FDG PET data of Parkinson's Disease patients and normal controls using our in-house software to derive a characteristic covariance pattern biomarker of disease.
Medicine, Issue 76, Neurobiology, Neuroscience, Anatomy, Physiology, Molecular Biology, Basal Ganglia Diseases, Parkinsonian Disorders, Parkinson Disease, Movement Disorders, Neurodegenerative Diseases, PCA, SSM, PET, imaging biomarkers, functional brain imaging, multivariate spatial covariance analysis, global normalization, differential diagnosis, PD, brain, imaging, clinical techniques
Community-based Adapted Tango Dancing for Individuals with Parkinson's Disease and Older Adults
Institutions: Emory University School of Medicine, Brigham and Woman‘s Hospital and Massachusetts General Hospital.
Adapted tango dancing improves mobility and balance in older adults and additional populations with balance impairments. It is composed of very simple step elements. Adapted tango involves movement initiation and cessation, multi-directional perturbations, varied speeds and rhythms. Focus on foot placement, whole body coordination, and attention to partner, path of movement, and aesthetics likely underlie adapted tango’s demonstrated efficacy for improving mobility and balance. In this paper, we describe the methodology to disseminate the adapted tango teaching methods to dance instructor trainees and to implement the adapted tango by the trainees in the community for older adults and individuals with Parkinson’s Disease (PD). Efficacy in improving mobility (measured with the Timed Up and Go, Tandem stance, Berg Balance Scale, Gait Speed and 30 sec chair stand), safety and fidelity of the program is maximized through targeted instructor and volunteer training and a structured detailed syllabus outlining class practices and progression.
Behavior, Issue 94, Dance, tango, balance, pedagogy, dissemination, exercise, older adults, Parkinson's Disease, mobility impairments, falls
Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients
Institutions: Hadassah Hebrew-University Medical Center.
In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to assess changes in myelination at the central nervous system (CNS). However, standard measurements, including routine visual tests and MRI scans, are not sensitive enough for this purpose. We present two visual tests addressing dynamic monocular and binocular functions which may closely associate with the extent of myelination along visual pathways. These include Object From Motion (OFM) extraction and Time-constrained stereo protocols. In the OFM test, an array of dots compose an object, by moving the dots within the image rightward while moving the dots outside the image leftward or vice versa. The dot pattern generates a camouflaged object that cannot be detected when the dots are stationary or moving as a whole. Importantly, object recognition is critically dependent on motion perception. In the Time-constrained Stereo protocol, spatially disparate images are presented for a limited length of time, challenging binocular 3-dimensional integration in time. Both tests are appropriate for clinical usage and provide a simple, yet powerful, way to identify and quantify processes of demyelination and remyelination along visual pathways. These protocols may be efficient to diagnose and follow optic neuritis and multiple sclerosis patients.
In the diagnostic process, these protocols may reveal visual deficits that cannot be identified via current standard visual measurements. Moreover, these protocols sensitively identify the basis of the currently unexplained continued visual complaints of patients following recovery of visual acuity. In the longitudinal follow up course, the protocols can be used as a sensitive marker of demyelinating and remyelinating processes along time. These protocols may therefore be used to evaluate the efficacy of current and evolving therapeutic strategies, targeting myelination of the CNS.
Medicine, Issue 86, Optic neuritis, visual impairment, dynamic visual functions, motion perception, stereopsis, demyelination, remyelination
Microsurgical Clip Obliteration of Middle Cerebral Aneurysm Using Intraoperative Flow Assessment
Institutions: Havard Medical School, Massachusetts General Hospital.
Cerebral aneurysms are abnormal widening or ballooning of a localized segment of an intracranial blood vessel. Surgical clipping is an important treatment for aneurysms which attempts to exclude blood from flowing into the aneurysmal segment of the vessel while preserving blood flow in a normal fashion. Improper clip placement may result in residual aneurysm with the potential for subsequent aneurysm rupture or partial or full occlusion of distal arteries resulting in cerebral infarction. Here we describe the use of an ultrasonic flow probe to provide quantitative evaluation of arterial flow before and after microsurgical clip placement at the base of a middle cerebral artery aneurysm. This information helps ensure adequate aneurysm reconstruction with preservation of normal distal blood flow.
Medicine, Issue 31, Aneurysm, intraoperative, brain, surgery, surgical clipping, blood flow, aneurysmal segment, ultrasonic flow probe