Name: a preclinical model to assess brain recovery after acute stroke in rats
Uploaded: 2019-11-06T13:00:00.000+00:00
Duration: 10 min 15 s
Description: Watch this Scientific Journal Video about A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats at JoVE.com

This work was supported by the National Natural Science Foundation of China (81603315, 81603316), Key R &#38; D plan of Jiangxi province in China (20171ACH80001), Industrial and academic cooperation projects in colleges and universities of Fujian province in China (2018Y41010011).

The procedure and use of animal subjects have been approved by the National Institute of Health for the care and use of laboratory animals. This protocol is specifically adjusted for the tests of middle cerebral artery occlusion/reperfusion (MCAO/R) and sensorimotor function.
1. Experimental design and grouping
<ol>
	<li>Use a rat MCAO/R model to screen a rat brain ischemic model method with more severe brain injury and greater model success ratio using Longa&#39;s score and TTC staining.
	<ol>
		<li>Perform the experiment on male Sprague-Dawley rats weighing 260&#8722;330 g that are 7&#8722;9 weeks of age. The real rat weight is 275 &#177; 15 g for 275 g, 300 &#177; 10 g for 300 g, and 320 &#177; 10 g for 320 g.</li>
		<li>Use the following seven groupings (weight, thread bolt type, infarct time): group 1 with 15 rats (275 g, 2636, 2 h); group 2 with 15 rats (275 g, 2636, 3 h); group 3 with 15 rats (275 g, 2838, 2 h); group 4 with 15 rats (275 g, 2838, 3 h); group 5 with 13 rats (300 g, 3040, 3 h); group 6 with 10 rats (320 g, 3040, 3 h); group 7 with 13 rats (300 g, 3043, 3 h).</li>
	</ol>
	</li>
	<li>Study the brain recovery status by TTC staining, and use suitable sensorimotor function tests to indicate the long-term functional deficits by the bilateral asymmetry test, grid-walking test, rotarod test and lifting rope test after 1, 35, 60, 90 days of MCAO/R.
	<ol>
		<li>Use male Sprague-Dawley rats weighing 300 &#177; 10 g that are 8&#8722;9 weeks of age.</li>
		<li>Use the following five groupings: a control (normal) group with 20 rats; a 1 day group with 16 rats; a 35 day group with 16 rats; a 60 day group with 17 rats; and a 90 day group with 19 rats.</li>
	</ol>
	</li>
	<li>After the Longa&#39;s score in step 1.1 or sensorimotor functional tests in step 1.2, anaesthetize and decapitate all rats for TTC staining.</li>
</ol>
2. Establishment of a unilateral MCAO/R model in rats9
NOTE: During the operation, use the microforceps gently to prevent breakage of the blood vessel. Avoid damage to the nerves and other blood vessels in the neck of the rat when the vessel is isolated. Care must be taken to present appropriate aseptic technique for all survival surgical procedures. The technique illustrated later in the video should be practiced through the entire procedure.
<ol>
	<li>Throughout the surgery, maintain the body temperature of the rats at 37.0 &#177; 0.5 &#176;C in a small animal thermostat. Prepare four 6 cm 5-0 sutures.</li>
	<li>Set the oxygen flow rate of a small animal anesthesia machine (with a waste gas treatment device) at 0.4&#8722;0.6 L/min and the concentration of isoflurane to be 5%. Place the rat in the anesthesia machine.</li>
	<li>After the animal has fainted, place the rat on a surgical fixing table. Connect the mouth of the rat to the mask of the anesthesia machine (the oxygen flow rate remains unchanged; adjust the concentration of isoflurane to be 3%). Confirm that the animal has entered deep anesthesia by observing a lack of extremity tension, corneal reflexes, and pain.</li>
	<li>Fix its limbs to lie on the operating table with paper bandages (or other tools).</li>
	<li>Remove the neck coat with an electric shaver and sterilize with 75% alcohol (iodophor is better). Fix the mouth of the rat with a hook.</li>
	<li>Cut 2&#8722;3 cm along the central longitudinal shape of the neck with ophthalmic scissors.</li>
	<li>Separate the common carotid artery. Separate the subcutaneous muscle with ophthalmic forceps. Use homemade retractor to fully expose the field of vision. After separating the anterior muscle of the trachea with ophthalmic forceps, separate along the right sternocleidomastoid tendon until the common carotid artery is visible.</li>
	<li>Isolate the common carotid artery, the external carotid artery and the internal carotid artery with ophthalmic forceps. Ligate the common carotid artery (hard knot), external carotid artery far from the heart end (hard knot), and internal carotid artery (loose knot) with 5-0 sutures. Line the external carotid artery near the heart end with 5-0 sutures.</li>
	<li>Insert a thread bolt. Cut a small opening in the external carotid artery using ophthalmic scissors and gently insert a thread bolt. Ligate the suture of the external carotid artery that has been in loose knot and cut off the external carotid artery.
	<ol>
		<li>Loosen the loose knot of the internal carotid artery and continue inserting the thread bolt to the beginning of the middle cerebral artery (suture marked). Then cut off the exposed thread bolt.</li>
	</ol>
	</li>
	<li>After the ischemic time is reached (2-3 h), fix the fracture of the external carotid artery with one microforceps, and gently pull out the thread bolt with another microforceps. When the front end of the thread bolt is completely withdrawn from the internal carotid artery, ligate the external carotid artery that was lined with 5-0 sutures, and then pull out the thread bolt completely.</li>
	<li>Loosen the common carotid artery, and daub ~50,000 U of penicillin sodium powder on the surface of the wound to prevent infection. Suture subcutaneous muscles and skin with 4 sutures.</li>
	<li>Give ~0.2 mL of sterile saline to the rat orally using a 1 mL syringe (SQ-PEN injection is better) to prevent postoperative water shortage after placing the rat back to the cage.</li>
	<li>Choose the animals after 24 hours of reperfusion according to the Longa&#39;s score10. Select animals with a Longa&#39;s score of 1&#8722;3 for the next TTC staining in step 1.1, and animals with a Longa&#39;s score of 2&#8722;3 score for the 1, 35, 60, 90 days study in step 1.2. 
	NOTE: Longa&#39;s score10: 0 score, no neurological deficit; 1 score, failure to extend left foreclaw; 2 score, circling to the left; 3 score, falling to the left; 4 score, cannot walk spontaneously and has a depressed consciousness.</li>
	<li>Analyze the Longa&#39;s score by one-way ANOVA. Values shown represent mean &#177; S.D. P &#60; 0.05 indicate difference.</li>
</ol>
3. TTC staining
NOTE: The rat brain slice mold and blade must be pre-cooled in a -20 &#176;C refrigerator before use to prevent adhesion caused by a large temperature difference. During staining, prevent adhesion between the brain slices and the culture plate, which can result in insufficient staining.
<ol>
	<li>Anesthetize the rat by intraperitoneal injection of 400 mg/kg chloral hydrate after the Longa&#39;s score in step 1.1 or sensorimotor functional tests in step 1.2.</li>
	<li>Decapitate the rat with surgical scissors or with a rat decapitation apparatus. Remove the brain with surgical scissors and hemostatic forceps.</li>
	<li>Put the brain in the refrigerator at -20 &#176;C for 30 min to facilitate slicing.</li>
	<li>Remove the brain from the refrigerator and place it in pre-cooled rat brain slice mold. Cut the brain into six 2-mm-thick consecutive sections with a pre-cooled blade.</li>
	<li>Stain the sections with 2% 5-triphenyl-2H-tetrazolium chloride (TTC) in a 6-well culture plate.</li>
	<li>Culture the sections for 30&#8722;60 min at 37 &#176;C in a shaking bed. Flip the sections every 10 min until the brain ischemia area and the normal area are clearly white and red.</li>
	<li>Line the brain slices vertically in order from the back to the front of brain. Use a ruler to ensure that the total length of each line is same. Take pictures with digital camera.</li>
	<li>Analyze the infarct volume.
	<ol>
		<li>Pre-treatment the photo with photoshop software
		<ol>
			<li>Import the photo using photoshop CS6. 00:00-00:14</li>
			<li>Click Select to select the brain slices, click Select | Inverse. 00:15-00:36</li>
			<li>Click Foreground to select black and click OK. 00:37-00:42</li>
			<li>Press Alt +Delete to fill the background color, and CTRL +D to deselect. 00:43-00:46</li>
			<li>Click File | Save to desktop. 00:47-01:08</li>
		</ol>
		</li>
		<li>Pre-treatment the photo with Image Pro Plus software.
		<ol>
			<li>Open Image Pro Plus 6.0 software and import the photo. 01:09-01:24</li>
			<li>For defect modification, adjust the brightness with the Contrast Enhancement tool, so that the background is black. 01:25-01:37</li>
			<li>Use the Median tool in Filter to remove the highlights. 01:38-01:46</li>
		</ol>
		</li>
		<li>Calculate the left (normal) brain area with Image Pro Plus software
		<ol>
			<li>Select color using Segmentation and adjust the value of H/S/I, so that the brain slices are separated from the black background. 01:47-02:12</li>
			<li>Return to Count | Size. 02:13-02:16</li>
			<li>Click on Count | Split Objects in Edit to separate the brain from the midline. The software will automatically distinguish the left and right brain areas. 02:17-02:49</li>
		</ol>
		</li>
		<li>Calculate the right infarct brain area with Image Pro Plus software
		<ol>
			<li>Implement step 3.8.1-3.8.2. 02:50-03:14</li>
			<li>Select Count | Size. 03:15-03:21</li>
			<li>Click on Draw | Merge Objects tool in Edit. Manually select the ischemic area and click Count to calculate the ischemic area. 03:16-05:31</li>
		</ol>
		</li>
		<li>Calculate the health brain area with Image Pro Plus software
		<ol>
			<li>Implement step 3.8.1-3.8.2. 05:32-06:44</li>
			<li>Select color using Segmentation and adjust the value of H/S/I, so that the normal part of brain slices are separated from the black background. 06:45-07:10</li>
			<li>Return to Count | Size and click on Count to calculate this area. 07:11-07:21</li>
			<li>Click on Split Objects in Edit to separate the brain from the midline. The software will automatically distinguish the left and right brain areas. 07:22-08:08 &#8203;</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Calculate the infarct volume (%) and infarct and shrink volume (%): 
	 
	Infarct volume (%) = [right infarct area/(2 x left brain area)] x 100. 
	Infarct and shrink volume (%) = [(left brain area - right health brain area) / (2 x left brain area)] x 100. 
	 
	NOTE: The right brain is the injured part. The data were analyzed by one-way ANOVA. Values shown represent mean &#177; S.D. P &#60; 0.05 indicate difference.</li>
</ol>
4. Assessment of sensorimotor function
NOTE: Rats (300 g, 3040 thread bolt, 3 h brain infarct time) with a Longa&#39;s score of 2&#8722;3 were selected to perform the sensorimotor function experiments from 1-90 days. Keep quiet and do not disturb the animals during this period of study. The data were analyzed by two-way ANOVA. Values shown represent mean &#177; S.D. P &#60; 0.05 indicate difference.
<ol>
	<li>Bilateral asymmetry test11
	<ol>
		<li>Wrap paper tape (5 cm long, 0.8 cm wide) on the saphenous part of each foreclaw of a rat three times with equal pressure.</li>
		<li>For each rat, record the number of times each foreclaw is contacted and tape removed in 5 min with camera, including unaffected paw times and affected paw times.</li>
		<li>After 30 min, repeat steps 4.1.1 and 4.1.2 again.</li>
		<li>Calculate the average value of the sensorimotor bias (%): 
		Sensorimotor bias (%) = (unaffected paw times - affected paw times)/(unaffected paw times + affected paw times) x 100</li>
	</ol>
	</li>
	<li>Grid-walking test
	<ol>
		<li>Place the rat in the center of an elevated grid surface platform (area: 1 m2; height: 90 cm) with grid openings of 2.5 cm2.</li>
		<li>Push the rat&#39;s hips lightly to encourage the rat to traverse the grid surface.</li>
		<li>Record the number of foot faults made by the unaffected (right) and affected (left) limbs and the total step number in 1 min with camera.</li>
		<li>Calculate the error times: 
		Error times (%) = [unaffected (right) limb - affected (left) limb]/total step number x 100. 
		NOTE: Total number of steps below 20-step data were removed.</li>
	</ol>
	</li>
	<li>Rotarod test12,13
	<ol>
		<li>Set up the rat rotating bar fatigue apparatus (diameter 90 mm) of rats using the supporting software to a speed of 13 rpm over a 5 min period on the computer.</li>
		<li>Start the computer programs and place the rat on the rotarod rungs at the same time.</li>
		<li>End a trial if the rat falls off the rung or keeps walking for 5 min and record the rotating time.</li>
		<li>Have the rat rest for 30 min.</li>
		<li>Repeat steps 4.3.2&#8722;4.3.4 twice more and choose the maximum value to be the last rotating time.</li>
	</ol>
	</li>
	<li>Lifting rope test14
	<ol>
		<li>Place the lifting rope instrument (70 cm high; the rope is 0.2 cm in diameter and 40 cm long) on the desk.</li>
		<li>Have the rat grip the rope with its forelimbs and hang the rat.</li>
		<li>Record the time of hanging and calculate the scores. 
		NOTE: A score of 3: 0&#8722;2 s on the rope; A score of 2: 3&#8722;4 s on the rope; A score of 1: 5&#8722;6 s on the rope; A score of 0: more than 7 s on the rope.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Many models establishing methods and behavioral indicators that are well used in acute cerebral ischemia may not have significant changes in the recovery and sequela stages of brain ischemia16,17. However, the number of patients with brain ischemic in the recovery and sequela stages is the greatest. It is essential to select a suitable animal model for the recovery and sequela stages of ischemia stroke.
We use the MCAO/R model in rats ...

Brain ischemia is divided into three stages with different post-stroke indicators: the acute stage (within 1 week), the recovery stage (1 week to 6 months), and the sequelae stage (more than 6 months). Presently, most studies focus on the acute stage of brain ischemia because of its significant effect and multi-relative research models1,2,3. However, the recovery and sequelae stages of brain ischemia cannot be ignored due to their long-term complication of disabilities. Therefore, the purpose of this study is to explore a stable, reliable and relatively simple animal model to research the recovery and sequela stages of brain ischemia.
Among the many experimental brain ischemia models, we use middle cerebral artery occlusion (MCAO) via thread bolt insertion into the right middle cerebral artery (MCA). This model is similar to human stroke, which can produce larger infarct volumes, result in many behavioral disorders related to stroke, and can allow blood reperfusion (R) by removing the thread bolt4,5,6. MCAO/R is also considered the gold standard animal model of brain ischemia7. Furthermore, the severity of the brain injury depends on the diameter and the insertion length of the thread bolt, the duration of brain ischemia, and the animal weight (larger rats have bigger brains and thicker cerebral vessels)8. Therefore, by changing the thread bolt type, the infarct time, and the rat weight, a suitable model can be found for the recovery and sequela stages of brain ischemia in MCAO/R rats. To validate the rat model, we performed a 1-day, 35-day, 60-day, and 90-day study of the MCAO/R model using TTC staining and sensorimotor function experiments (a bilateral asymmetry test, a grid-walking test, a rotarod test and a lifting rope test).

<table><tbody><tr><td>Anatomical Microscope</td><td>Leica (Germany)</td><td>S8</td><td>Microscopic operating instrument</td></tr><tr><td>Blade</td><td>Gellette</td><td>/</td><td>Cutting brain sections</td></tr><tr><td>Constant Temperature Shaking Bed</td><td>Taicang Experimental Equipment Factory</td><td>THZ-C</td><td>To keep the brain sections stained evenly and at a constant temperature</td></tr><tr><td>Digital Camera</td><td>Canon</td><td>700D</td><td>For taking pictures of TTC staining</td></tr><tr><td>Electric Shaver</td><td>Shanghai Yuyan Scientific Instruments Co., Ltd.</td><td>3000#</td><td>Removal of hair from the neck of rats</td></tr><tr><td>Forceps Hamostatic</td><td>Shanghai Medical device Co., Ltd.</td><td>14 cm</td><td>Using for brain removing</td></tr><tr><td>Image Pro Plus Software</td><td>Media Cybernetics Inc.</td><td>6.0</td><td>Analyze the infarct volume</td></tr><tr><td>Isoflurane</td><td>RWD Life Science</td><td>217170702</td><td>Anesthetic gas</td></tr><tr><td>Microforceps</td><td>Shanghai Jinzhong Medical Devices Co., Ltd.</td><td>10 cm</td><td>Vascular micromanipulation</td></tr><tr><td>Microshear</td><td>Shanghai Jinzhong Medical Devices Co., Ltd.</td><td>10 cm</td><td>Vascular micromanipulation</td></tr><tr><td>Ophthalmic Forceps</td><td>Shanghai Jinzhong Medical Devices Co., Ltd.</td><td>10 cm</td><td>Auxiliary skin and muscle anatomy</td></tr><tr><td>Pphthalmic Scissors</td><td>Shanghai Jinzhong Medical Devices Co., Ltd.</td><td>10 cm</td><td>Using for cutting the skin of neck</td></tr><tr><td>Rat Brain Slice Mold</td><td>Shanghai Yuyan Scientific Instruments Co., Ltd.</td><td>400 g</td><td>For standard, uniform cutting of brain tissue</td></tr><tr><td>Rat Rotating Bar Fatigue Apparatus</td><td>Anhui Zhenghua Biological Instrument and Equipment Co., Ltd.</td><td>ZH-300B</td><td>To test the sensorimotor function</td></tr><tr><td>Small Animal Anaesthesia Machine</td><td>Shanghai Yuyan Scientific Instruments Co., Ltd.</td><td>ABM3000</td><td>A gas anesthetic machine</td></tr><tr><td>Small Animal Thermostat</td><td>Beijing Damida Technology Co., Ltd.</td><td>DM.7-YLS-20A</td><td>To maintain animal body temperature constant during operation</td></tr><tr><td>Surgical Scissors</td><td>Shanghai Medical device Co., Ltd.</td><td>16 cm</td><td>Using for decapitate and brain removing</td></tr><tr><td>Suture</td><td>Shanghai Jinhuan Medical Devices Co., Ltd.</td><td>4-0 / 5-0</td><td>Using for skin and muscle sutures / Using for vascular ligations</td></tr><tr><td>Thread Bolt</td><td>Beijing Cinontech Co. Ltd.</td><td>2636/2838/3040/3043-A4</td><td>Blockage of the middle cerebral artery in rats</td></tr><tr><td>5-triphenyl-2H-tetrazolium chloride (TTC)</td><td>Sigma</td><td>LOT#BCBP3272V</td><td>Brain section staining reagent</td></tr></tbody></table>

a preclinical model to assess brain recovery after acute stroke in rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

Using the abovementioned procedure for a MCAO/R model with a Longa&#39;s score and TTC staining, different treatments of average weight (275/300/320 g), bolt types (2636/2838/3040/3043; Table 1) and ischemic times (2-3 h) and 1 day reperfusion were used to screen for the optimal brain ischemia model in rats. Model parameters of 300 g weight, 3040 thread bolt, and 3 h brain infarct time were the most suitable for the largest cerebral infarction, highest Longa&#39;s score a...

Watch this Scientific Journal Video about A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats at JoVE.com

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

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Research

JoVE Journal

Neuroscience

8.2K Views.  Beijing Yinfeng Dingcheng Biological Engineering Technology Ltd.. The purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

Video: A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

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.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

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 ...

Detecting Behavioral Deficits in Rats After Traumatic Brain Injury

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, few studies have investigated the long-term effects of injury in rodent models of TBI. Shown here are behavioral measures that are well-established in TBI research for times early after injury, such as two weeks, until two months. Some of these methods have previously been used at later times after injury, up to one year, but by very few laboratories. The methods demonstrated here are a short neurological assessment to test reflexes, a Beam-Balance to test balance, a Beam-Walk to test balance and motor coordination, and a working memory version of the Morris water maze that can be sensitive to deficits in reference memory. Male rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid percussion injury (FPI) or sham injury. Rats can be tested on the short neurological assessment (neuroscore), the beam-balance, and the Beam-Walk multiple times, while testing on the water maze can only be done once. This difference is because rats can remember the task, thus confounding the results if repeated testing is attempted in the same animal. When testing from one to three days after injury, significant differences are detected in all three non-cognitive tasks. However, differences in the Beam-Walk task were not detectable at later time points (after 3 months). Deficits were detected at 3 months in the Beam-Balance and at 6 months in the neuroscore. Deficits in working memory were detected out to 12 months after injury, and a deficit in a reference memory first appeared at 12 months. Thus, standard behavioral tests can be useful measures of persistent behavioral deficits after FPI.

The goal of the behavioral tests presented here is to detect functional deficits in rats after traumatic brain injury. Four specific tests are presented that detect deficits in behaviors to reflect the damage to specific brain areas at times extending to one year after injury.

With the increasing incidence of traumatic brain injury (TBI) in both civilian and military populations, TBI is now considered a chronic disease; however, ...

Behavior

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Medicine

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

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 &#8220;coagulation model&#8221;. 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 &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and contributes significantly to the development of vascular dementia. Few models of focal white matter stroke exist and this lack of appropriate models has hampered understanding of the neurobiologic mechanisms involved in injury response and repair after this type of stroke. The main limitation of other subcortical stroke models is that they do not focally restrict the infarct to the white matter or have primarily been validated in non-murine species. This limits the ability to apply the wide variety of murine research tools to study the neurobiology of white matter stroke. Here we present a methodology for the reliable production of a focal stroke in murine white matter using a local injection of an irreversible eNOS inhibitor. We also present several variations on the general protocol including two unique stereotactic variations, retrograde neuronal tracing, as well as fresh tissue labeling and dissection that greatly expand the potential applications of this technique. These variations allow for multiple approaches to analyze the neurobiologic effects of this common and understudied form of stroke.

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

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 ...

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.

Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...