Name: transient middle cerebral artery occlusion model of neonatal stroke in p10 rats
Uploaded: 2017-04-21T12:30:00.000+00:00
Duration: 7 min 56 s
Description: Watch this Scientific Journal Video about Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats at JoVE.com

Funding was provided by the NIH K08 NS064094 and UCSF REAC grants. The authors would like to acknowledge Nikita Derguin, Zinalda Vexler, and Joel Faustino for their assistance in the development of this technique.

All animal research was approved by the University of California, San Francisco Committee on Animal Research and was performed in accordance with the Guide for the Care and Use of Laboratory Animals (US Department of Health and Human Services, Publication No. 85-23, 1985). Animals were closely monitored by veterinarians of the UCSF Institutional Animal Care and Use Committee (IACUC), accredited by AAALAC. One female Sprague-Dawley rat with an 8 day-old litter (10 pups per litter) was obtained. The mother and her pups were given food and water ad libitum and housed in a temperature- and light-controlled animal care facility with daily enrichment, per IACUC protocol, until the pups were 10 days old. All surgical instruments used in this procedure were autoclaved to ensure sterility. Sterility of instrument tips is maintained throughout the surgery.
1. Middle Cerebral Artery Occlusion
<ol>
	<li>Weigh the pup and ensure that it is the proper weight (19-21 g). Anesthetize the pup in 3% isoflurane in 100% O2 and ensure that there is no response to a foot pinch. Maintain the body surface temperature between 35.5 &#176;C and 37 &#176;C with the use of a heating pad under the surgical stage.</li>
	<li>Secure the animal in a supine position with tape across the shoulder regions. Using sterile cotton swabs, swab the anterior cervical area with povidone-iodine solution followed by a swab of 70% ethanol in double-distilled water, alternating between each solution for four swabs total.</li>
	<li>Locally infiltrate 0.25% bupivacaine into the planned incision site. Using a stereoscope, make a midline 5 to 7 mm anterior cervical incision to expose the common carotid artery (CCA). Place 2-4 retractors to keep the cavity open and the artery exposed.</li>
	<li>Locate the internal carotid artery (ICA), occipital artery (OA), and external carotid artery (ECA). Groom the arteries to get a clear view. Be careful not to disturb the vagus nerve. 
	NOTE: For sham-operated pups, the inicision is left open and the arteries are exposed, after which the incision is sutured closed. The total anesthesia time is equivalent to that of the occlusion surgery.</li>
	<li>Cut 1.5 cm of 6-0 silk braided suture thread. Unbraid the suture, pulling out single strands. Make sure that the single strands are neat and not frayed. 
	NOTE: If necessary, smoothe the frayed ends by dipping the single strand in sterile water and grooming the strand with forcep tips.</li>
	<li>While holding the suture strand with 45-degree forceps, move the forceps in a sweeping arc motion to go under the ICA so that the forcep tips emerge between the ICA and OA. 
	NOTE: If the dissection is done well, this step will be relatively easy. If the ICA and OA are touching, be careful not to rupture the arteries when using the forceps to isolate the ICA. If bleeding occurs, apply pressure to the artery with the forceps until the bleeding stops. Absorb the blood with a sterile swab.</li>
	<li>Grab the end of the suture strand that is held by the forceps and pull it so that the end is easy to access. Release the strand from the forceps and back the forceps out from under the ICA, reversing the motion in step 1.5 (Figure 1A).</li>
	<li>Tie a temporary ligature around the ICA at the base, closest to where it separates from the CCA. 
	NOTE: It is important to tie the knot so that the end of the strand that will be pulled to remove the knot is long enough (greater than 1 mm, less than 3 mm) to easily grasp with forceps, while ensuring that there is an appropriate amount of suture strand on the other side of the knot for retraction.</li>
	<li>Carefully retract the ICA laterally and use a clip to secure the strand to excess skin near the axilla region on the side opposite the incision. Ensure that this retracting strand is taut enough to stop blood flow before proceeding to the next step, to minimize the risk of uncontrolled bleeding. Observe that the artery is flat and pale. 
	NOTE: Do not over-retract, as it can cause a partial or full tear of the ICA. The retraction can be adjusted just before making the arteriotomy by pulling on the strand on the side opposite to the clip.</li>
	<li>Use 45&#176; forceps to grasp another unbraided suture strand and loop it under and around the ICA, as in step 1.5. Position this strand lateral to the retraction strand (Figure 1B). 
	NOTE: This step can also be done prior to retraction. If unsure about the quality of retraction, this knot can be very loosely tied before the next step.</li>
	<li>Cut a 0.2 mm arteriotomy midway between the tied and untied ligatures, erring closer to the tied ligature. 
	NOTE: Blood remaining in the tied-off artery may empty through the arteriotomy but should not exceed 5 &#181;L. If bleeding persists, carefully pull the retraction strand to increase retraction, with care to avoid damaging the artery from excessive tension.</li>
	<li>Using a metric ruler, measure the occluding suture and cut the suture with an extra allowance of 2-3 mm for the removal of the occluder during reperfusion. Hold the occluder with 45-degree forceps and use straight forceps to create a bend at the appropriate length to reach the MCA, marking a stopping point for advancement. 
	NOTE: A 10 mm occlusion length from the silicone tip end to the bend is used for P10 Sprague Dawley or Long Evans rat pups in this weight range.</li>
	<li>Using 45&#176; forceps, feed the silicone-coated nylon occlusion suture into the arteriotomy and advance the suture to the bend that marks the pre-determined distance to the MCA (Figure 1C). Ensure that advancement feels smooth; immediately stop advancement if resistance is felt. During advancement, aim the suture in a direction that is parallel to the CC/ECA, towards the head. 
	NOTE: If the suture is advanced dorsally, toward the animal&#39;s spine, it may run into the pterygopalatine artery (PTA). If resistance is felt after 3-5 mm of advancement, the suture has hit the PTA junction. Back the occluder out of the artery until the silicone head is near the arteriotomy before adjusting the advancement direction. It is not necessary to completely remove the occluder from the arteriotomy.</li>
	<li>Secure the occluder by tying a temporary ligature, using the strand from step 1.10 (Figure 1D).</li>
	<li>Remove the retractor clip. Trim the strands of both of the temporary ligatures so that the strand of the knot that is pulled to remove the knot is easy to grasp with straight forceps and is longer than the strand that is pulled to tighten the knot. 
	NOTE: The strands must be short enough so that they do not tangle in the cavity after closure.</li>
	<li>Remove the retractors and close the cavity using 6-0 braided silk to create three to four interrupted sutures.</li>
	<li>Remove the pup from anesthesia and place it on a heating pad in room air. Monitor the pup until it has regained sufficient consciousness to maintain sternal recumbency and ensure that it has fully recovered before returning it to the dam. Ensure that the pup maintains a body surface temperature between 35.5 &#176;C and 37 &#176;C.</li>
	<li>During occlusion, diffusion-weighted magnetic resonance imaging (DW-MRI) under isoflurane anesthesia can be used to verify the appropriate induction of injury. Figure 2 demonstrates ongoing ischemic injury detected by DW-MRI during tMCAO9. 
	NOTE: Diffusion-weighted spin echo planar imaging is performed 10-15 min prior to reperfusion. The entire brain is imaged with serial 2-mm-thick coronal sections using the following pulse sequence settings: TR/TE=5,000/60 ms, 4 averages, field of view = 35 mm, data matrix = 128x128, diffusion gradient duration = 20 ms, separation = 29.7 ms, amplitude = 70mT/m, and b-factor = 1,045 s/mm2. Animals exhibiting a lack of cortical involvement or atypical ischemic injury, such as in the brainstem, are excluded.</li>
</ol>
2. Reperfusion
NOTE: Occlusion is performed for 3 h to cause a moderate-to-severe amount of injury involving the striatum and cortex.
<ol>
	<li>At approximately 2 h and 50 min following occlusion of the MCA, anesthetize the pup in 3% isoflurane in 100% O2. Maintain a body surface temperature between 35.5 &#176;C and 37 &#176;C with the use of a heating pad under the surgical stage.</li>
	<li>Remove the interrupted sutures from step 1.16 and locate the junction, which is marked by the two ligatures and the tail end of the occluding suture. 
	NOTE: Sham-operated animals are induced and remain under anesthesia for an equivalent time period to the occluded animals. The incision is once again opened and sutured closed. For sham-operated animals, proceed to step 2.11.</li>
	<li>Carefully untie the most lateral knot previously tied in step 1.14 by pulling the longer strand. 
	NOTE: If there is resistance in untying the knot, stop to ensure that the correct strand of the knot is being pulled. If resistance continues, increase magnification for a better view. Be cautious when untying, as it is possible to damage the artery during this step.</li>
	<li>Remove the suture strand from the cavity.</li>
	<li>At exactly 3 h following MCA occlusion, slowly back the occluding suture out of the artery. There will be no resistance. 
	NOTE: In most cases, there will be no bleeding. If a small amount of bleeding occurs, apply pressure to the artery at the arteriotomy site.</li>
	<li>Use forceps to apply pressure to the arteriotomy, as the next step restores ICA blood flow for reperfusion.</li>
	<li>Carefully untie the medial knot using the same method as in step 2.3.</li>
	<li>Remove the suture strand from the body and apply a hemostatic agent to the arteriotomy to stop bleeding.</li>
	<li>Ensure that the bleeding has stopped. 
	NOTE: Return of the artery&#39;s original shape and red color confirms that ICA blood flow has been restored.</li>
	<li>Remove the retractors and close the cavity with three to four interrupted sutures of 6-0 braided silk.</li>
	<li>Remove the pup from anesthesia and place it on a heating pad in room air. Monitor it until it has regained sufficient consciousness to maintain sternal recumbency and ensure full recovery before returning it to the dam. Ensure that the pup maintains a body surface temperature between 35.5 &#176;C and 37 &#176;C.</li>
	<li>Inspect the pups daily for 5 days. Recprd their weights and inspect the incision sites closely for appropriate healing. Remove the sutures after 7-14 days. Post-tMCAO injury can be imaged with MRI, if desired9. 
	NOTE: Animals may lose up to 1 g of weight on the first day but will typically regain the weight without intervention and be within a similar weight range to controls by days 4-5. Rarely, a pup may lose excessive weight or have difficulty feeding, requiring oral gavage feeds for 2-3 days.</li>
	<li>At P21, injury can be reliably assessed with sensorimotor behavioral tests, such as rotarod or cylinder rearing. Cognitive testing can be performed as early as 4-6 weeks of age using assessments such as novel object recognition or the Morris water maze13.</li>
	<li>Euthanize the rats by intraperitoneal injection of Euthasol (50 mg/kg) for brain harvest11,12.</li>
	<li>Histological analysis to assess injury volume or response to interventions can be performed with cresyl violet or H&#38;E staining (Figure 3)10.</li>
</ol>

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The authors have nothing to disclose.

Critical steps within the protocol
First, it is important to maintain normothermia from the initiation of anesthesia until full recovery, as there are known effects of both hypothermia17 and hyperthermia18 on the progression of brain injury in both immature and mature animals. Second, while securing the animal and retracting the incision, optimal positioning to monitor breathing and to ensure that the trachea is free of compression is essential. Third, avoid squeezing or stretching the vagus nerve, as this may cause changes in heart rate with vagal stimulation. Fourth, because retraction of the ICA is necessary to control bleeding during the arteriotomy, attention must be paid to the degree of tension during retraction to avoid damaging the artery. If the artery does tear from retraction, or if there is a poor arteriotomy incision, the animal should be excluded from analysis due to the risk of hemorrhage and poor reperfusion.
Modifications and troubleshooting
Using MRI as a guide, the suture length may be optimized to ensure that the silicone tip properly occludes the MCA to create the focal ischemia. If MRI is not available, pups may be euthanized before reperfusion for dissection to visualize the placement of the suture. Adjust the suture length as needed. The pup weight highly correlates with the occluding suture length requirements. The occlusion time can be modified to adjust the degree of injury severity.
In addition, suture shape and length are critical. For P10 Sprague-Dawley and Long Evans rats weighing 19-21 g, 10 mm is the optimal length of insertion in our experience. Further insertion of the occluding suture may result in perforation of the MCA. Furthermore, the consistency in the shape of the occluding filament in each surgery will result in an increased consistency of injury pattern19,20. For this reason, we recommended using professionally-manufactured sutures for this specific purpose. It is also important to note that the injury pattern may differ between practitioners due to seemingly minute differences in technique.
Limitations of the technique
Performing this technique in a small, developing rodent requires significant experience. If performed correctly, the surgeon is able to cause a very consistent injury pattern across animals of different sizes and attain a survival rate greater than 95%. Furthermore, proper surgical tools are essential. Surgical instruments must be well maintained to ensure that all instrument tips approximate properly.
Significance of this technique with respect to existing or alternative methods
While hypoxia-ischemia, or the Rice-Vannucci model2, is most commonly used to study hypoxic-ischemic injury in the developing brain, it is important to note that this model of tMCAO is distinct from HI in that there is transient focal ischemia without global hypoxia, followed by a reperfusion phase when the obstruction is removed and blood flow is restored. This causes a more consistent and reproducible injury and is more clinically translational by causing an injury pattern similar to that seen in full-term neonatal stroke. This enables the study of focal injury patterns and compensatory responses in uninjured tissue.
Future applications after mastering this technique
This model is similar to the most common cause of stroke in human neonates, a transient occlusive thrombus that occurs during the perinatal period11,21. The etiology is not entirely clear and is most likely multifactorial, but it is presumed in most cases to result from emboli passing from the placenta11. In addition, many newborns with presumed perinatal stroke often present with later seizure activity or subtle focal neurological exam abnormalities22. This makes the use of a consistent, translational injury model to identify mechanisms of injury progression and possible therapeutic strategies crucial.

Stroke during the neonatal period is a significant cause of death and disability, occurring in as many as 1 in 2,300 live births1. This leads to altered central nervous system development and increased long-term morbidity, including increased incidence of epilepsy, cerebral palsy, mental retardation, and other types of motor or cognitive dysfunction. The lifelong effects of early stroke make translational animal models essential for examining the mechanisms of injury and repair in this population, including strategies to protect the injured brain or to enhance repair.
Different ischemia models have been used to study brain injury in adult animals, and while the Rice-Vannucci (Modified Levine)2 procedure is commonly used to study hypoxic-ischemic injury in the developing brain, focal ischemia-reperfusion is a distinct mechanism of injury causing focal injury, with an injured core and penumbra and uninjured remote tissue. The Koizumi3 and Longa4 models were developed in adult rats to achieve transient middle cerebral artery occlusion via the common carotid artery (CCA) and external carotid artery (ECA), respectively. In both models, permanent ligation and cauterization of artery branches are important to minimize bleeding and to streamline the surgical procedure, which also causes adverse effects on the animal&#39;s ability to feed and to gain weight following injury. Furthermore, there are distinct injury mechanisms in the immature brain and specific patterns of injury seen as a result.
More recently, photothrombotic stroke (Rose-Bengal method)5 and permanent MCA ligation6 have been used to study neonatal and adult stroke. Both photothrombotic stroke and MCA ligation create permanent changes in cerebral blood flow that result in a lack of reperfusion. Reperfusion is a critical component of the development and progression of focal injury, with increased excitotoxicity, free radical formation, and nitric oxide production leading to delayed cell death that involves signaling cascades that are distinct from the ischemic phase7. Hypoxia-ischemia involves permanent unilateral carotid ligation followed by global hypoxia, which also differs from the cause of hypoxic-ischemic injury in humans and does not cause a consistent focal injury pattern, making study of the injured core and penumbra more challenging.
We have previously described a non-hemorrhagic ischemia-reperfusion stroke model in the immature rat using transient middle cerebral artery occlusion (MCAO)8,9,10. This is a less invasive method that accesses and occludes the MCA through the internal carotid artery without permanent ligation or cauterization. This provides a model of injury similar to the most common cause of stroke in the perinatal period11,12. This ischemia-reperfusion model of injury results in damage to the ipsilateral striatum and parieto-temporal cortex. This model of tMCAO also allows control over the severity of injury by varying the duration of occlusion. Examination of signaling pathways and histological changes in the injured core and penumbra and in the uninjured ipsilateral and contralateral tissue can further elucidate the mechanisms of injury and repair in the immature brain. This study will demonstrate this important injury model for the developing brain.

<table><tbody><tr><td>Isoflourane</td><td>Henry Schein</td><td>50033</td><td>anesthetic, at 3%&amp;nbsp;</td></tr><tr><td>Trinocular Surgioscope</td><td>World Precision Instruments</td><td>PSMT5N</td><td></td></tr><tr><td>Heating pad</td><td>Sunbeam</td><td>000731-500-000</td><td>low to medium setting</td></tr><tr><td>IR Thermometer</td><td>Extech Instruments</td><td>72-5270</td><td></td></tr><tr><td>Retraction kit for small animals&amp;nbsp;</td><td>Fine Science Tools</td><td>18200-20</td><td></td></tr><tr><td>CermaCut Scissors</td><td>Fine Science Tools</td><td>14958-09</td><td></td></tr><tr><td>Dumont #5SF Forceps</td><td>Fine Science Tools</td><td>112522-00</td><td>2x</td></tr><tr><td>Dumont #5/45 Forceps</td><td>Fine Science Tools</td><td>11251-35</td><td>2x</td></tr><tr><td>B-2 Micro Clamp</td><td>Fine Science Tools</td><td>00398-02</td><td></td></tr><tr><td>Forcepts for Clamp Application</td><td>Fine Science Tools</td><td>00072-14</td><td></td></tr><tr><td>Micro Vannas Scissors</td><td>Fine Science Tools</td><td>15000-03</td><td>2mm cutting edge</td></tr><tr><td>Occlusion Sutures</td><td>Doccol</td><td>602123PK10</td><td>701712PK5Re</td></tr><tr><td>Ruler</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Hemostatic Agent&amp;nbsp;</td><td>Avitene</td><td>DVL1010590</td><td></td></tr><tr><td>6-0 Perma-Hand Silk Reverse CuttingSuture</td><td>Ethicon</td><td>769G</td><td></td></tr><tr><td>Euthasol</td><td>Virbac</td><td>710101</td><td>0.22 mL/kg</td></tr><tr><td>Cotton Tipped Applicators</td><td>Henry Schein</td><td>100-9249</td><td></td></tr><tr><td>Laboratory Tape</td><td>VWR</td><td>89097-990</td><td></td></tr></tbody></table>

transient middle cerebral artery occlusion model of neonatal stroke in p10 rats

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

The severity of injury caused by tMCAO is highly dependent upon both the occlusion time and the experience of the surgeon. A 90-min occlusion often produces a mild to moderate injury pattern, while 3 h produces a moderate-to-severe injury. Severity of injury may be assessed through a variety of methods, including MRI, histology, or short- or long-term behavioral analyses. Figure 2 demonstrates an example of the DW-MRI performed 75 min into a 90-min occlusion, confirming ischemic injury involving the ipsilateral hemisphere. Diffusion weighted imaging demonstrates increased diffusion in the ipsilateral striatum and the majority of the ipsilateral cortex, without contralateral changes, during the acute ischemic phase. This correlates to a moderate level of long-term injury involving both the cortex and the deep gray matter.
Occlusion of the MCA results in cell death that begins in the striatum for less severe injury and develops worsening cortical and hippocampal injury for longer occlusion times and more severe injury. During optimization of the surgical technique, such as determining the suture insertion length, MRI is highly recommended, as it allows for the confirmation of proper suture placement and visualization of edema and injury progression during occlusion14,15,16. If MRI is not available, H&#38;E or cresyl violet staining are simple and reliable histological methods to determine injury morphology and can be used at both early and late time points after the tMCAO. Figure 3 demonstrates a moderate-to-severe injury pattern on histopathological examination following a 3 h occlusion, demonstrating cyst formation and volume in the ipsilateral striatum and cortex.
Unbiased stereological quantification of cresyl-violet-stained sections to calculate the ratio of ipsilateral to contralateral hemispheric volume confirms ipsilateral ischemic-reperfusion injury. Specifically, after anesthetizing the animal with Euthasol, the brain was harvested by transcardiac perfusion in 0.1 M PBS with 4% paraformaldehyde. Following overnight postfixation and equilibration in 30% sucrose, the entire brain was sectioned at 50 &#181;m intervals, and every twelfth section was selected, mounted, and stained with cresyl violet10.
Even with mild injury, locomotor changes, such as circling and hemiparesis, are noted during the occlusion period. With more severe injury, these changes will persist after reperfusion. Additional behavioral testing can be used to assess the severity of injury, including rotarod or cylinder rearing testing for sensorimotor function and the Morris water maze for cognitive function13.
<img alt="Figure 1" src="/files/ftp_upload/54830/54830fig1.jpg" /> 
Figure 1: Live Surgical Images of the tMCAO Procedure. (A) The first suture strand is looped around the ICA, as detailed in step 1.6. (B) The first temporary ligature is tied and the ICA is retracted. The second suture strand is looped around the ICA, lateral to the first suture strand, as detailed in step 1.9. (C) The silicone coated occlusion suture is fed into the arteriotomy site, as detailed in step 1.12. (D) The second temporary ligature is tied to secure the occluder in place, as detailed in step 1.14. Scale bar = 1 mm.
<img alt="Figure 2" src="/files/ftp_upload/54830/54830fig2.jpg" /> 
Figure 2: MRI During Occlusion Demonstrates the Appropriate Unilateral Injury. Anterior to posterior, coronal image slices of the DW-MRI, performed during a 90 min occlusion, demonstrate increased diffusion involving the ipsilateral hemisphere (arrows), which is consistent with ongoing ischemic injury in the acute phase. Reprinted with permission from Stroke11. <a href="http://cloudflare.jove.com/files/ftp_upload/54830/54830fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54830/54830fig3.jpg" /> 
Figure 3: Unilateral Injury Involving the Striatum and Cortex at 4 weeks following tMCAO. Posterior to anterior, cresyl-violet-stained coronal brain sections (each 50 &#181;m) harvested from P38 animals demonstrate fairly severe injury (the arrows show ipsilateral cyst formation and reduced cortical and striatal volume) following a 3 h tMCAO at P10. The round hole on the left side represents a contralateral hemisphere identifier. Scale bar = 5 mm. Reprinted with permission from Neurobiology of Disease12. <a href="http://cloudflare.jove.com/files/ftp_upload/54830/54830fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats at JoVE.com

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

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

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

transient-middle-cerebral-artery-occlusion-model-neonatal-stroke-p10

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.5K Views.  University of California, San Francisco. Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

Video: Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 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 ...

Neuroscience

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

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

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

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 Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

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 ...

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 ...

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. ...

A Model of Transient Cerebral Ischemia by Occlusion of Middle Cerebral Artery

Transient Middle Cerebral Artery Occlusion Model of Stroke

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models.
In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

Author Spotlight: Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model 

This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies ...

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (&gt;12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term ...

Middle Cerebral Artery Occlusion in Rats with Preserved Vascular Anatomy

A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion Without Damaging the Anatomical Structure of Cerebral Vessels

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have significantly contributed to our understanding of its pathological mechanisms and the development of potential treatments. Presently, there are two common methods involving filament (endovascular suture) techniques to induce animal models of cerebral ischemia. However, these methods have inherent limitations, such as reduced blood perfusion to the brain, damage to the external carotid artery system, impaired food and/or water intake, and sensory dysfunction of the face. This article introduces a new method for inducing a rat ischemic stroke model without compromising the cerebral vascular anatomy. In this study, the common carotid artery (CCA) of Sprague-Dawley rats was exposed, and an incision was made. A filament was then inserted through the incision into the internal carotid artery to occlude the middle cerebral artery. After 1.5 h of induced ischemia, the occluding filament was fully removed from both the internal carotid artery and the CCA. The incision in the CCA was subsequently sutured using 11-0 microsurgical sutures under a microscope (magnification 4x). Through the utilization of microsurgical techniques to repair the CCA, this study successfully developed a unique method to induce an ischemic stroke model in rats while preserving the anatomical integrity of cerebral blood vessels.

The current protocol describes a rat model of middle cerebral artery occlusion/reperfusion that preserves the anatomical structure of cerebral vessels without causing damage.