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

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 ...

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 ...

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

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 ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

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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 ...

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 ...

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 ...

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

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 ...

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 ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...