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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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...

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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

8.1K 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

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume three-dimensionally. While MRI is an extremely sensitive tool for detecting tissue abnormalities, association of signal changes with an underlying pathological process is usually not straightforward. In the central nervous system, for example, inflammation, demyelination, axonal damage, gliosis, and neuronal death may all induce similar findings on MRI. As such, interpretation of MRI scans depends on the context, and radiological-histopathological correlation is therefore of the utmost importance. Unfortunately, traditional pathological sectioning of brain tissue is often imprecise and inconsistent, thus complicating the comparison between histology sections and MRI. This article presents novel methodology for accurately sectioning primate brain tissues and thus allowing precise matching between histology and MRI. The detailed protocol described in this article will assist investigators in applying this method, which relies on the creation of 3D printed brain slicers. Slightly modified, it can be easily implemented for brains of other species, including humans.

The overall goal of this protocol is to accurately align magnetic resonance imaging (MRI) image volumes with histology sections via the creation of customized 3D-printed brain holders and slicer boxes.

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

Motor Dual-Tasks for Gait Analysis and Evaluation in Post-Stroke Patients

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask gait analysis consisted of a single walking task (Task 0), a simple motor dual-task (water-holding, Task 1), and a complex motor dual-task (crossing obstacles, Task 2). The task of crossing obstacles was considered to be equivalent to the combination of a simple walking task and a complex motor task as it involved more nervous system, skeletal movement, and cognitive resources. To eliminate heterogeneity in the results of the gait analysis of the stroke patients, the dual-task gait cost values were calculated for various kinematic parameters. The major differences were observed in the proximal joint angles, especially in the angles of the trunk, pelvis, and hip joints, which were significantly larger in the dual motor tasks than in the single walking task. This research protocol aims to provide a basis for the clinical diagnosis of gait function and an in-depth study of motor control in stroke patients with motor control deficits through the analyses of dual-motor walking tasks.

This paper presents a protocol specifically for dual motor task gait analysis in stroke patients with motor control deficits.

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask ...

Dural Stimulation and Periorbital von Frey Testing in Mice As a Preclinical Model of Headache

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system. For example, they protect the brain from the skull and anchor/organize the vascular and neuronal supply of the cortex. However, the meninges are also implicated in nervous system disorders such as migraine, where the pain experienced during a migraine is attributed to local sterile inflammation and subsequent activation of local nociceptive afferents. Of the layers in the meninges, the dura mater is of particular interest in the pathophysiology of migraines. It is highly vascularized, harbors local nociceptive neurons, and is home to a diverse array of resident cells such as immune cells. Subtle changes in the local meningeal microenvironment may lead to activation and sensitization of dural perivascular nociceptors, thus leading to migraine pain. Studies have sought to address how dural afferents become activated/sensitized by using either in vivo electrophysiology, imaging techniques, or behavioral models, but these commonly require very invasive surgeries. This protocol presents a method for comparatively non-invasive application of compounds on the dura mater in mice and a suitable method for measuring headache-like tactile sensitivity using periorbital von Frey testing following dural stimulation. This method maintains the integrity of the dura and skull and reduces confounding effects from invasive techniques by injecting substances through a 0.65 mm modified cannula at the junction of unfused sagittal and lambdoid sutures. This preclinical model will allow researchers to investigate a wide range of dural stimuli and their role in the pathological progression of migraine, such as nociceptor activation, immune cell activation, vascular changes, and pain behaviors, all while maintaining injury-free conditions to the skull and meninges.

The most notable symptom of migraine is severe head pain, and it is hypothesized that this is mediated by sensory neurons innervating the meninges. Here, we present a method to locally apply substances to the dura in a minimally invasive manner while using facial hypersensitivity as an output.

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system.  ...

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Author Spotlight: Innovative Techniques and Future Directions in Stroke Research 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...