Name: assessing neuroprotective effects of glycyrrhizae radix et rhizoma extract using a transient middle cerebral artery occlusion mouse model
Uploaded: 2018-12-09T13:00:00
Description: Watch this Scientific Journal Video about Assessing Neuroprotective Effects of Glycyrrhizae Radix et Rhizoma Extract Using a Transient Middle Cerebral Artery Occlusion Mouse Model at JoVE.com

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All procedures involving animals were approved by the ethics committee of Pusan National University (approval number, PNU-2016-1087). A graphical overview of this study is shown in Figure 1.
1. Preparation and Administration of GRex
NOTE: The GR used in this study was purchased from a commercial pharmaceutical company.
<ol>
	<li>Place 200 g of GR in 2,000 mL of methanol and incubate at room temperature (25 &#176;C) for 5 days.</li>
	<...

<ol>
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With the increasing prevalence of metabolic diseases such as chronic hypertension, diabetes, and hyperlipidemia, which are major risk factors for stroke, stroke prevention and treatment have become an important area of medical research12,13. Deficits in language and movement after a stroke are strongly correlated with the degree of damage to brain tissue14 and result in a poor quality of life for patients and their families<sup class="xref...

Brain damage caused by ischemia and reperfusion of cerebral blood flow leads to the death of nerve cells and loss of brain tissue. This type of brain damage continues to increase with the increasing prevalence of cerebrovascular diseases due to the spread of metabolic diseases such as obesity, hypertension, and diabetes mellitus1,2. The absolute number of elderly patients with stroke has dramatically increased worldwide, and the cost of medical care for these patients, who are often left with long-term disabilities, is a major societal burden. Therefore, secondary disabilities should be mitigated as much as possible to reduce the economic burden1,2.
The most commonly used rodent model of cerebral infarction is the middle cerebral artery (MCA) occlusion (MCAO) model, in which the MCA is occluded with a silicon-coated surgical suturing filament to block blood flow, causing ischemic stroke3,4. Using a filament as the occlusion material allows the control of occlusion time and permanence by manipulating the duration of the intra-luminal filament insertion.
Previous studies have shown that even when the same rodent MCAO model is used, the total volume of cerebral infarction varies between experiments, causing low reproducibility of the studies. To improve reproducibility, we optimized the thickness of the filament mint used in the experiment. The results of a preliminary study of the cerebral ischemic period and induced infarction showed that an ischemic period longer than 60 min allowed the volumetric region of damaged brain tissue to be observed and quantified.
Glycyrrhizae Radix et Rhizoma (GR), also known as licorice, consists of the dried roots and rhizomes of Glycyrrhiza uralensis and G.&#160;glabra. It has been used in Chinese and Korean traditional medicine for various purposes including as a food additive and medicinally5,6,7.
In a previous study8, pre-treatment with GR methanol extract (GRex) showed an anti-apoptotic effect in MCAO mice, including significant prevention of the decrease in the protein expression of B-cell lymphoma 2 (Bcl-2) and Bcl extra-large (Bcl-xL). This study was conducted to improve the reproducibility of the conventional MCAO mouse model by evaluating its efficiency in determining if post-infarct treatment with GRex effectively reduced the infarct volume in MCAO-induced cerebral damage.

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			<td>Sigma</td>
			<td>CLS431220</td>
			<td>Syringe filter (0.45 &#181;m)</td>
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			<td height="40" style="height:40px;">Stereo Microscope</td>
			<td>Leica</td>
			<td>M50</td>
			<td>Stereo Microscope</td>
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			<td height="40" style="height:40px;">Stereo Microscope</td>
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			<td>Stereo Microscope</td>
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			<td>Carl Zeiss</td>
			<td>Zeiss Axio</td>
			<td>Microscope</td>
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			<td height="40" style="height:40px;">Data Analysis</td>
			<td>Systat Software Inc.</td>
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			<td>Doo Yeol Biotech</td>
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			<td>Nylon suture (8-0 monofilament)&#160;</td>
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			<td>2,3,5-triphenyltetrazolium chloride (TTC)</td>
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			<td>JUNSEI</td>
			<td>69360-1263 20KG</td>
			<td>Formalin (Formaldehyde solution)</td>
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			<td>EasyStain</td>
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			<td>Sigma</td>
			<td>C5042-10G</td>
			<td>Cresyl violet (acetate)</td>
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			<td>Sigma-Aldrich</td>
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			<td>Moria Pascheff-Wolff Spring Scissors</td>
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			<td>18153-11</td>
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assessing neuroprotective effects of glycyrrhizae radix et rhizoma extract using a transient middle cerebral artery occlusion mouse model

Ischemia followed by reperfusion of cerebral blood flow after a stroke leads to the death of nerve cells and loss of brain tissue. The most commonly used animal model for studying stroke is the middle cerebral artery occlusion (MCAO) model. Previous research studies have reported different infarct sizes even when the same experimental animal species was used under similar MCAO conditions. Therefore, we developed an improved experimental method to address this discrepancy. Mice were subjected to MCAO using a filament as the occlusion material to mimic human stroke conditions and filament thickness was optimized to establish more reproducible infarction volume. Mice treated with a methanol extract of Glycyrrhizae Radix et Rhizome (GRex) following stroke induction showed a significantly decreased total infarction volume and increased number of surviving cells relative to the untreated control group. This modified experimental protocol successfully and reproducibly demonstrated the beneficial effect of GRex on ischemic stroke.

In the sham-operated normal group, no cerebral infarct is observed whereas in the control group, a relatively wide range of damaged areas is observed. In the mice administered 300 mg/kg GRex in the MCAO model group, a statistically significant reduction in damaged area is observed (Figure 2).
The histological changes are investigated by staining ischemic brain sections with H&#38;E or cresyl violet....

Watch this Scientific Journal Video about Assessing Neuroprotective Effects of Glycyrrhizae Radix et Rhizoma Extract Using a Transient Middle Cerebral Artery Occlusion Mouse Model at JoVE.com

Assessing Neuroprotective Effects of Glycyrrhizae Radix et Rhizoma Extract Using a Transient Middle Cerebral Artery Occlusion Mouse Model

In this study, we modify an existing experimental method to obtain more reproducible results, by establishing a middle cerebral artery occlusion (MCAO) mouse model. Oral administration of Glycyrrhizae Radix et Rhizome (GR) methanol extract (GRex), following stroke induction, significantly decreased total infarction volume relative to the untreated control group.

Ischemia followed by reperfusion of cerebral blood flow after a stroke leads to the death of nerve cells and loss of brain tissue. The most commonly used ...

This method can help answer key questions in the neuroscience and traditional oral medicine research fields about screening for neuroprotective natural compounds relevant to the Model. The main advantage of this technique is that the higher producibility of the infection volume is in accordance with ischemic timing dose. Visual demonstration of this method is critical as it is typical to insert a very thin implement into the original, the middle cerebellar artery using a sterile microscope.Demonstrating the procedure will be Se-Eun Lee, a grade a school student studying in my laboratory. To prepare the Glycyrrhizae Radix et Rhizome extract, or GRex, first add 200-grams of Glycyrrhizae Radix et Rhizome, or GR, to two-liters of methanol for a five day incubation at room temperature. At the end of the incubation, strain the solution through 0.26-millimeter thick filter paper with a five-micrometer pore size.Add the GR residue to one-liter of fresh methanol, and filter the solution again. Combine the two filtrates, and strain them through a new piece of filter paper. Then concentrate the extract under vacuum before freeze-drying to produce GRex.To prepare the GRex for administration, dissolve the freeze-dried product in dimethylsulfoxide, and dilute the mixture with 0.9%physiological saline. Then filter the solution through a 0.45-micrometer syringe filter, and adjust the final concentration of DMSO to less than 5%To set up a mouse middle cerebral artery occlusion model, or MCAO, confirm a lack of responsive to tail stimulus in an anesthetized six-week old 22 to 25-gram male C57BL/6 mouse, and place the mouse on a 36.5-degree Celsius body temperature holding blanket, connected to a thermometer. After removing all the hair from the chest and neck of the mouse by shaving and depilatory cream, place the mouse under a stereomicroscope and make a two-centimeter skin incision in the center of the neck.Carefully isolate the left common carotid artery, external carotid artery, and the branch of the internal carotid artery from the surrounding connective tissues, and use a 4.0-silk suture to ligate the external carotid artery and the common carotid artery with a half-hitch knot to temporarily block the bloodflow into the internal carotid artery. Insert a 0.1 to 0.12-millimeter thick 11-millimeter long silicone-coated 8.0-monofilament 4.0-silk suture through the internal carotid artery to the origin of the left middle cerebral artery, and use a laser Doppler flow meter to measure the decrease in relative cerebral bloodflow artery. Fix the inserted filament to the blood vessel for two hours while the cerebral artery is occluded.At the end of the occlusion period, carefully withdraw the filament to restore the bloodflow for 22 hours of reperfusion. And use 3.0-silk sutures to suture the skin in five places with two half hitches knots. Then return the mouse to its cage for anesthetic recovery.One hour after the end of reperfusion, administer 300-milligrams per kilogram body weight GRex to the experimental animals, only by oral gavage. 24 Hours after the onset of MCAO, make an incision in the middle skin of the head of each experimental animal, and break the parietal bones with angled forceps, while peeling off the dura mater. Carefully isolate the brain from the skull, and use a mouse brain matrix to cut the excised tissue into one-millimeter thick sections.Stain the sections for 17 minutes in 2%TTC solution, according to standard protocols. And fix the sections in 10%formalin for at least 2 hours before imaging the samples with a digital camera. Then analyze and quantify the cerebral infarct area of each section using ImageJ.At the appropriate experimental time point post-MCAO, perfuse each euthanized experimental animal transcardially with 10-milliliters of PBS, followed by 10-milliliters of 4%paraformaldehyde. After isolating the brains as just demonstrated, immerse the harvested organs in 10-milliliters of 30%sucrose overnight. The next morning, embed the brain tissue samples in optimal cutting temperature compound.And slice the tissues coronally into 15-micrometer thick sections on a cyrostat. Mount the sections on glass slides and submerge the sections in 80%ethanol for 1 minute. For H&E staining, label the samples with Hematoxylin solution for 5 minutes, followed by two dips in 1%acid alcohol.Next, immerse the slides in saturated Lithium carbonate solution for 30 seconds, followed by a 30-second wash in tap water, and 30 seconds of counter-staining with Eosin solution. Remove the excess Eosin solution with a tap water wash, and dehydrate the slides with one-minute ascending immersions in 95 then 100%ethanol. Then, airdry the slides, clear them in xylene for at least 10 minutes, and mount the coverslips with mounting medium.For cresyl violet staining, place the 80%ethanol-treated slides on a slide warmer for at least one hour, followed by immersion in 50%ethanol diluted with chloroform overnight. The next morning, stain the slides with 0.1%cresyl violet for 10 minutes in a 40-degree Celsius dry oven, followed by a dehydration in 95%ethanol for 30 minutes, and two five-minute 100%ethanol immersions. Next, clear the slides two five-minute xylene immersions, followed by air drying.Then mount the slides with mounting medium for visualization by light microscopy. In sham-operated normal groups, no cerebral infarct is observed;whereas in control, untreated MCAO animals, a relatively wide range of damaged areas is observed. In 300-milligram per kilogram GRex-treated model mice, a statistically significant reduction in damaged tissue is observed.Histological analysis of the H&E and cresyl violet-stained tissue sections provides an index of cell survival. Indeed, H&E and cresyl violet staining intensities significantly decrease in the control, untreated MCAO group relative to the sham-operated normal group. The GRex-treated animals, on the other hand, exhibit a greater histological integrity, implying less neuronal cell death than both the control, untreated and sham-operated groups, suggesting a potent neuroprotective effect of the extract against ischemia reperfusion-induced brain injury.While attempting this procedure, it's important to remember to monitor the relative cerebellar bloodflow during this period. And conductors of processes very carefully to minimize damages to the endothelial layers of the

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Research

JoVE Journal

Medicine

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

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.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Mouse Model of Middle Cerebral Artery Occlusion

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.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

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 ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

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.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

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. in 19972. 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.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...