Name: a modified transcranial middle cerebral artery occlusion model to study stroke outcomes in aged mice
Uploaded: 2023-05-05T14:50:00.000+00:00
Duration: 4 min 46 s
Description: Watch this Scientific Journal Video about Innovative Techniques and Future Directions in Stroke Research at JoVE.com

The authors thank Kathy Gage for her editorial support. Scheme figures were created with BioRender.com. This study was supported by funds from the Department of Anesthesiology (Duke University Medical Center) and NIH grants (NS099590, HL157354, NS117973, and NS127163).

All procedures described in this work are conducted in accordance with the NIH guidelines for the care and use of animals in research, and the protocol was approved by the Duke Institute Animal Care and Use Committee (IACUC). Young (8-10 weeks old) and aged (22 months old) male C57Bl/6 mice were used for the present study. An overview of this protocol is illustrated in Figure 1.
1. Surgical preparation
<ol>
	<li>Examine the mouse for gross abnormalities and behavioral deficits. 
	NOTE: Before surgery, it&#39;s important for surgeons to wear appropriate PPE (protective personal equipment), including surgical mask, cap, gloves, and gown.</li>
	<li>Weigh the mouse; program the ventilator (see Table of Materials) based on the body weight.</li>
	<li>Place the mouse in a 4 in x 4 in x 7 in anesthesia induction box. Turn on the oxygen flow meter (see Table of Materials), set at 30, and the nitrous oxide flow meter, set at 70. Turn on the vaporizer with 5% isoflurane.</li>
	<li>Insert the guide wire into the 20 G intravenous (IV) catheter.</li>
	<li>Take the mouse out of the induction box when its respiratory rate is reduced to 30-40 breaths per minute.</li>
	<li>Lay the mouse on the surgical bench in a supine position. Pull the mouse&#39;s tongue out and hold it with the fingers of the left hand. Insert a laryngoscope (see Table of Materials) into the animal&#39;s mouth to visualize the vocal cord.</li>
	<li>Stabilize the mouse chin on the laryngoscope using the right middle finger. Free the left hand to hold the 20 G IV catheter.</li>
	<li>Insert the guide wire slightly into the vocal cord, and then slowly push the 20 G IV catheter into the trachea until the wing part of the catheter becomes even with the nose tip. 
	NOTE: If the mouse is moving, do not insert the wire. This may cause trauma to the trachea and bleeding.</li>
	<li>Turn on the ventilator (see Table of Materials), and connect it with the 20 G IV catheter intubated in the mouse. Reduce the isoflurane to 1.5%, and ensure that both lungs are mechanically ventilated. 
	&#8203;NOTE: Do not forget to reduce the isoflurane concentration. Otherwise, the mouse will receive an overdose of anesthesia.</li>
	<li>Apply eye ointment onto both eyes and inject 5 mg/kg carprofen subcutaneously.</li>
	<li>Keep the mouse in a lateral position with the right temporal area facing up. Maintain the rectal temperature at 37 &#176;C using a heating pad (35 &#176;C) and a heat lamp controlled by a temperature regulator (see Table of Materials).</li>
	<li>Shave the surface area between the right eye and ear, and disinfect the surgical area at least three times with iodine and alcohol swabs.</li>
</ol>
2. MCAO surgery
<ol>
	<li>Open the sterile instrument package for MCAO surgery. Wear sterile gloves and make a 1 cm skin incision between the right eye and right ear using surgical scissors. 
	NOTE: Monitor skin color, body temperature, and response to toe pinch every 15 min.</li>
	<li>Dissect the underlying fascia with forceps to expose the temporal and masseter muscles. 
	NOTE: Be careful not to damage the parotid gland.</li>
	<li>Use forceps to touch the lower part of the temporal muscle and detect the location of the zygomatic arch. Carefully pull aside the branches of the facial nerve.</li>
	<li>Use the tip of a high-temperature cautery loop (see Table of Materials) to cut a 5 mm transverse incision on the temporal muscle.</li>
	<li>Use two forceps to dissect the underlying zygomatic arch and expose the joint between the maxilla and zygomatic bones.</li>
	<li>Use scissors to cut a 3 mm portion of the zygomatic arch and remove it. Separate the masseter muscle from the skull base. 
	NOTE: Be careful not to fracture the retro-orbital sinus and superficial temporal vein.</li>
	<li>Apply four small retractors positioned in different directions to expose the cranial skull base, with the trigeminal nerve branches pulled laterally by one retractor. 
	NOTE: A sulcus on the external surface of the cranial base marks the location of the lateral fissure between the frontal and temporal lobes. The MCA lies here (Figure 2A), and its trunk and branches are visible through the thin, transparent skull (Figure 2B). The relationship of this artery to other major cerebral arteries is displayed in Figure 2A.</li>
	<li>Apply a drop of 0.9% normal saline on the skull above the MCA trunk and proximal to the branch of the rhinal cortex. Use an electrical grinder to thin the skull until a small fracture is visible. 
	NOTE: Do not push the grinder against the skull, as it may penetrate the skull and injure the underlying artery.</li>
	<li>Use the tip of the forceps to lift the thinned skull and remove it. For sham mice, stop here, and do not ligate the artery. 
	NOTE: A small rectangular window across the MCA trunk is formed.</li>
	<li>Place a single-strand loop of black braided silk on top of the MCA (Figure 2C). Insert an 8-0 microsurgical needle to lift the MCA trunk, and tie the suture (see Table of Materials) under the needle, leaving both ends of the needle on the top of the silk thread loop knot (Figure 2D).</li>
	<li>For transient MCAO, tighten the silk thread knot slightly under the needle to block arterial blood flow (Figure 2E), representing MCAO onset.</li>
	<li>Use the forceps to hold the suture, and slowly remove the needle at the end of the ischemia (e.g., 60 min or longer). 
	NOTE: When the needle is removed, the silk thread knot is slipped off the MCA, and the brain is reperfused (Figure 2F).</li>
	<li>For permanent MCAO, firmly tighten the silk thread loop around the artery, and remove the needle. Cut and remove the excess suture materials.</li>
	<li>Apply a drop of 0.25% bupivacaine to the skin incision, and suture the muscle and skin separately using 6-0 nylon sutures intermittently (see Table of Materials). Apply antibiotic ointment to the surface of the skin incision. 
	NOTE: The skin incision can also be closed with sterile staples or glue.</li>
</ol>
3. Post-surgical care
<ol>
	<li>Turn the isoflurane off to awaken the mouse. Disconnect the ventilator when spontaneous respiration is restored.</li>
	<li>Transfer the mouse to a recovery chamber (see Table of Materials) with a controlled temperature.</li>
	<li>Extubate the mouse when its righting reflex is restored, or it begins to move.</li>
	<li>Closely monitor the mouse in a temperature- and humidity-controlled chamber. Return the mouse to the home cage after it gains full consciousness (recovery period ~2 h). Administer 5 mg/kg Carprofen subcutaneously daily for 3 days.</li>
</ol>
4. Laser speckle contrast imaging (LSCI)
<ol>
	<li>Six and 24 h post-MCAO, mount the anesthetized mouse on the stereotaxic frame. Shave the top of the head, and clean it with three alternating swabs of iodine and alcohol. 
	NOTE: Anesthetization was performed as mentioned in step 1.3. LSCI is also performed before MCAO.</li>
	<li>Make a 3 cm midline skin incision and dissect the skin from the skull. Apply four small needle retractors to expose the skull top.</li>
	<li>Move the laser speckle camera (see Table of Materials) above the head, and adjust the focus of the camera. Image the cerebral blood flow.</li>
</ol>
5. 2,3,5-triphenyltetrazolium chloride (TTC) staining
<ol>
	<li>Deeply anesthetize the mouse with 5% isoflurane at the end of the experiment, typically on day 1, 3, or 28 after stroke. Pinch the tail to ensure there is no pain response.</li>
	<li>Decapitate the mouse using a surgical scissor and harvest the brain. Incubate the brain in ice-cold saline for 20 min.</li>
	<li>Put the brain in a brain slicer matrix on ice and drop cold saline on the brain. Cut the brain into 1 mm slices using thin razor blades.</li>
	<li>Immerse the brain slices at the same orientation into a dish of 2% TTC solution (see Table of Materials). Keep the dish in the dark at room temperature for 15 min. 
	NOTE: Normal brain tissue becomes red and ischemic tissue remains white.</li>
	<li>Transfer the brain slices to 10% formalin for 24 h of fixation. Image the brain slices and measure the infarct area.</li>
</ol>

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

<ol>
	<li>Tsao, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics-2022+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics-2022 update: a report from the American Heart Association.</a> Circulation. 145 (8), e153 (2022).</li><li>Nogueira, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombectomy+6+to+24+hours+after+stroke+with+a+mismatch+between+deficit+and+infarct.">Thrombectomy 6 to 24 hours after stroke with a mismatch between deficit and infarct.</a> The New England Journal of Medicine. 378 (1), 11-21 (2018).</li><li>Fisher, M., Savitz, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+brain+cytoprotection+in+acute+ischaemic+stroke-renewed+hope+in+the+reperfusion+era.">Pharmacological brain cytoprotection in acute ischaemic stroke-renewed hope in the reperfusion era.</a> Nature Reviews Neurology. 18 (4), 193-202 (2022).</li><li>Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20 (1), 84-91 (1989).</li><li>Knauss, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+semiquantitative+non-invasive+measurement+of+PcomA+patency+in+C57BL/6+mice+explains+variance+in+ischemic+brain+damage+in+filament+MCAo.">A semiquantitative non-invasive measurement of PcomA patency in C57BL/6 mice explains variance in ischemic brain damage in filament MCAo.</a> Frontiers in Neuroscience. 14, 576741 (2020).</li><li>Yang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-ischemia+common+carotid+artery+occlusion+worsens+memory+loss,+but+not+sensorimotor+deficits,+in+long-term+survived+stroke+mice.">Post-ischemia common carotid artery occlusion worsens memory loss, but not sensorimotor deficits, in long-term survived stroke mice.</a> Brain Research Bulletin. 183, 153-161 (2022).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+O-GlcNAcylation+is+neuroprotective+in+young+and+aged+brains+after+ischemic+stroke.">Increasing O-GlcNAcylation is neuroprotective in young and aged brains after ischemic stroke.</a> Experimental Neurology. 339, 113646 (2021).</li><li>Jiang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XBP1+(X-box-binding+protein-1)-dependent+O-GlcNAcylation+Is+neuroprotective+in+ischemic+stroke+in+young+mice+and+its+impairment+in+aged+mice+is+rescued+by+thiamet-G.">XBP1 (X-box-binding protein-1)-dependent O-GlcNAcylation Is neuroprotective in ischemic stroke in young mice and its impairment in aged mice is rescued by thiamet-G.</a> Stroke. 48 (6), 1646-1654 (2017).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptomic+analysis+of+the+immune+cell+landscape+in+the+aged+mouse+brain+after+ischemic+stroke.">Single-cell transcriptomic analysis of the immune cell landscape in the aged mouse brain after ischemic stroke.</a> Journal of Neuroinflammation. 19 (1), 83 (2022).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+effects+of+neuronal+ATF6+activation+in+permanent+ischemic+stroke.">Beneficial effects of neuronal ATF6 activation in permanent ischemic stroke.</a> Frontiers in Cellular Neuroscience. 16, 1016391 (2022).</li><li>Tamura, A., Graham, D. I., McCulloch, J., Teasdale, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+cerebral+ischaemia+in+the+rat:+2.+Regional+cerebral+blood+flow+determined+by+[14C]iodoantipyrine+autoradiography+following+middle+cerebral+artery+occlusion.">Focal cerebral ischaemia in the rat: 2. Regional cerebral blood flow determined by [14C]iodoantipyrine autoradiography following middle cerebral artery occlusion.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 1 (1), 61-69 (1981).</li><li>Tamura, A., Graham, D. I., McCulloch, J., Teasdale, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+cerebral+ischaemia+in+the+rat:+1.+Description+of+technique+and+early+neuropathological+consequences+following+middle+cerebral+artery+occlusion.">Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 1 (1), 53-60 (1981).</li></ol>

All authors have no conflict of interest.

The first transcranial MCA occlusion model was established in rats in 198111,12, and replaced by the no-craniectomy MCAO model in 19894. The initial transcranial MCA occlusion had a wide surgical field, such that the entire zygomatic arch was removed and the muscles pulled laterally. Local tissues were swollen after surgery, causing stress and decreased food intake for the animals. In our modified transcranial MCAO model, the incision is less invasive, and only a small segment of the zygomatic arch is removed. The surgical field is exposed using four small needle retractors, and no blood vessels or nerves are destructed. A small skull window is sufficient because the MCA trunk is lifted using an 8-0 surgical suture needle, and the entire needle does not need to go under the MCA. No local tissue swelling was found after surgery6.
This model has several advantages. First, it produces an infarct area that includes both cortex and sub-cortex regions, and thus, neurologic deficits can be readily assessed. Second, both transient and permanent ischemic stroke can be induced in this model. Importantly, an extended ischemic duration can be applied to mimic late reperfusion. For example, in our previous stroke study, a 6 h MCAO was successfully performed9. Third, reliance on the PcomA for collateral blood supply and reperfusion is minimal, which reduces the variability of stroke severity. Finally, almost all mice, even aged mice, can survive long-term functional studies. Taken together, this model exhibits excellent clinical relevance.
Of note, this stroke model has limitations. First, a high level of microsurgical skill is required. A novice animal surgeon may need some time to perfect craniotomy and MCA ligation under a stereomicroscope. Careful execution of grinding, skull removal, and suture placement is key to successfully implementing this model. Moreover, ligating the MCA at the same location for each animal is critical. Second, the meninges are slightly damaged by the needle in this model, which may need to be considered for studies focused on the meninges. Lastly, although an ischemic duration &#62;6 h may be performed, reperfusion must be confirmed by measuring cerebral blood flow with laser Doppler or laser speckle imaging.
In summary, this modified mouse stroke model induces moderate brain damage, enables long-term survival experiments to be performed in aged and stroke comorbidity animals, and is expected to advance experimental stroke research and novel drug development to improve stroke outcomes.

Nearly 800,000 people suffer a stroke in the US every year, and most of these strokes are ischemic in nature1. Timely restoration of the cerebral blood flow with tissue plasminogen activator (tPA) and/or thrombectomy is currently the most effective treatment for stroke patients; however, the full recovery of neurologic functions in the long term is rare2,3. Thus, searching for novel stroke therapy that targets functional improvement is an intense area of research that requires clinically relevant animal models of stroke. 
The most common ischemic stroke model in rodents uses intraluminal middle cerebral artery occlusion (MCAO) to induce stroke. In this model, initially developed by Zea Longa in 1989, a nylon filament is introduced into the internal carotid artery (ICA) to block the blood flow to the middle cerebral artery (MCA)4. However, this model has limitations. First, when the filament is inserted into the ICA, the blood flow to the posterior cerebral artery (PCA) could be partially blocked as well, especially in mice. Critically, the posterior communicating artery (PcomA), a small artery that connects anterior and posterior cerebral circulation, is frequently underdeveloped in some mouse strains, such as C57Bl/6, the strain predominantly used in experimental stroke research. This patency of the PcomA is believed to contribute to the variability in lesion size in mice after stroke5. Indeed, when blood flow to the PCA drops precipitously during MCAO, and the PcomA is unable to provide sufficient collateral blood flow, the stroke infarct can expand into the territory of the PCA. Moreover, in this model, a long duration of ischemia leads to a higher chance of mortality in mice. Consequently, a short MCAO duration of 30-60 min is typically used in mice. However, most stroke patients experience a few hours of ischemia before reperfusion treatment. Thus, a mouse stroke model with an extended duration of ischemia is of high clinical relevance. 
The overall goal of this procedure is to model ischemic stroke in mice that have a medium-sized infarct and an excellent survival rate. This transcranial MCAO model addresses critical attributes of clinical stroke, as prolonged ischemia can be performed, and aged mice tolerate this model well, allowing for the long-term assessment of functional recovery.

<table><tbody><tr><td>0.25% bupivacaine</td><td>Hospira</td><td>NDC 0409-1159-18</td><td></td></tr><tr><td>0.9% sodium chloride</td><td>ICU Medical</td><td>NDC 0990-7983-03</td><td></td></tr><tr><td>2,3,5-Triphenyltetrazolium Chloride (TTC)&amp;nbsp;</td><td>Sigma or any available vendor</td><td></td><td></td></tr><tr><td>20 G IV catheter</td><td>BD</td><td>381534</td><td>20 GA 1.6 IN</td></tr><tr><td>30 G needle</td><td>BD</td><td>305106</td><td></td></tr><tr><td>4-0 silk suture</td><td>Look</td><td>SP116</td><td>Black braided silk</td></tr><tr><td>8-0 suture with needle&amp;nbsp;</td><td>Ethilon</td><td>2822G</td><td></td></tr><tr><td>Alcohol swabs</td><td>BD</td><td>326895</td><td></td></tr><tr><td>Anesthesia induction box</td><td>Any suitable vendor</td><td></td><td>Pexiglass make&amp;nbsp;</td></tr><tr><td>Electrical grinder</td><td>JSDA</td><td>JD 700</td><td></td></tr><tr><td>High temperature cautery loop tip</td><td>Bovie</td><td>AA03</td><td></td></tr><tr><td>Isoflurane</td><td>Covetrus</td><td>NDC 11695-6777-2</td><td></td></tr><tr><td>Laser doppler perfusion monitor</td><td>Moor Instruments</td><td>moorVMS-LDF1</td><td></td></tr><tr><td>Lubricant eye ointment</td><td>Bausch + Lomb</td><td>339081</td><td></td></tr><tr><td>Mouse rectal probe</td><td>Physitemp</td><td>RET-3</td><td></td></tr><tr><td>Nitrous Oxide</td><td>Airgas</td><td>UN1070</td><td></td></tr><tr><td>Otoscope</td><td>Welchallyn</td><td>728</td><td>2.5 mm Speculum Otoscope served as a laryngoscope to visualize vocal cords in mice</td></tr><tr><td>Oxygen</td><td>Airgas</td><td>UN1072</td><td></td></tr><tr><td>Povidone-iodine</td><td>CVS</td><td>955338</td><td></td></tr><tr><td>Recovery box</td><td>Brinsea&amp;nbsp;</td><td>TLC eco</td><td></td></tr><tr><td>Rimadyl (carprofen)</td><td>Zoetis</td><td>6100701</td><td>Injectable 50 mg/mL</td></tr><tr><td>Rodent ventilator</td><td>Kent Scientific</td><td>Rodent Jr.</td><td></td></tr><tr><td>Temperature controller</td><td>Physitemp</td><td>TCAT-2DF&amp;nbsp;</td><td></td></tr><tr><td>Triple antibioric &amp;amp; pain relief</td><td>CVS</td><td>NDC 59770-823-56</td><td></td></tr><tr><td>Vaporizer</td><td>RWD</td><td>R583S</td><td></td></tr></tbody></table>

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.

With a direct view under a surgical microscope, it can be visually confirmed that MCA blood flow is blocked during ischemia. Our previous study showed a &gt;80% blood flow reduction in the ischemic area using a laser Doppler monitor6. In order to determine post-MCAO blood flow changes, LSCI can be used to further confirm the ischemic insult and reperfusion (Figure 1). Indeed, in Figure 3A, it is observed that the blood supply was reduced in the territory of the right MCA. For transient MCAO, after the suture was removed, reperfusion of the cerebral blood flow was evident (Figure 3B), and was further improved 24 h later (Figure 3C). The stroke brain can be sectioned after 24 h and stained with TTC. Dead tissue did not react with TTC, and remained white (Figure 1). TTC staining demonstrated that this model generates infarcted tissue in both the cortical and the lateral striatum areas, and that the infarct size is moderate compared to filament MCAO (Figure 4). This model has been applied to young and aged animals, and a negligible mortality rate (&lt;5%) was found over 28 days of observation7.
This model causes motor and sensory deficits, predominantly in the left front paw. Our previous studies show neurologic deficits in stroke mice, as evidenced by various behavioral tests such as the cylinder test, open field test, tape removal test, pole test, and Von Frey filament test6,8,9,10. Mice subjected to 90 min of transcranial MCAO also demonstrate cognitive deficits compared to sham-operated mice6. Although the long-term functional outcome after transcranial MCAO has not been systemically examined in aged mice, a similar model in aged rats clearly showed neurologic deficits over 28 days after stroke7.
<img alt="Rodent surgical window; infarct analysis via TTC staining; LSCI imaging for cerebral blood flow." class="xfigimg" src="/files/ftp_upload/65345/65345fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Overview of the protocol. The right MCA is transiently or permanently occluded through a small skull window in mice. TTC staining and LSCI are used to determine the infarct size and evaluate post-ischemia cerebral blood flow, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65345/65345fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Brain occlusion procedure diagram with labeled arteries, surgical images of occlusion site." class="xfigimg" src="/files/ftp_upload/65345/65345fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: The steps of transcranial MCAO surgery. (A) Location of the ligated MCA. (B) Exposure of the MCA trunk and its branches. (C) A single strand of a silk suture is placed above the MCA. (D) An 8-0 needle is used to lift the MCA trunk, and the suture is tied under the needle. (E) The suture is slightly tightened to block the blood flow. (F) The needle and suture are removed to allow reperfusion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65345/65345fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Thermal imaging of cerebral blood flow, showing heat maps in brain vessels; color-coded diagram." class="xfigimg" src="/files/ftp_upload/65345/65345fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Laser speckle contrast images in MCAO with delayed reperfusion. (A) The right hemisphere had a low perfusion area (red arrow), indicating ischemia. (B) After 6 h of ischemia, the suture was removed to allow reperfusion, and the arterial branches became visible. (C) After 24 h, blood flow perfusion was improved in these arterial branches. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65345/65345fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Brain blood vessel anatomy; microscope image; panel of brain cross-sections; red and white tissue." class="xfigimg" src="/files/ftp_upload/65345/65345fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Difference from the filament MCAO. (A) The ink-perfused brain shows the blood vessels on the brain surface. The red arrow points to the MCA trunk, which is ligated in this transcranial MCAO model. The green arrow points to the MCA origin, which is the site of MCA occlusion in the filament MCAO model. The brain infarct is visible at 24 h post-stroke on the TTC-stained brain slides. The samples here are from (B) 60 min of filament MCAO in a young mouse, and (C) permanent transcranial MCAO in young (8-10 weeks old), and (D) aged C57Bl/6 mice (22 months old). Normal tissue is red, and infarcted tissue is white. The infarct size in this model is moderate, and the infarcted area includes both the cortex and striatum. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65345/65345fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Innovative Techniques and Future Directions in Stroke Research at JoVE.com

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.

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. ...

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3.5K Views.  Duke University Medical Center. Beroerte treft jaarlijks bijna 800.000 mensen in de VS. Ongeveer 87% hiervan is ischemisch, wat zich presenteert als een ernstige medische aandoening. Er zijn echter beperkte behandelingsopties beschikbaar, vooral tijdens de fase van chronische beroerte. Ons onderzoek is gericht op het ontwikkelen van een herstelbevorderende therapie om invaliditeit te verminderen en de kwaliteit van leven van patiënten met een beroerte te verbeteren.Ze gebruiken het filament, het occlusiemodel van de...

Video: Auteur in de schijnwerpers: innovatieve technieken en toekomstige richtingen in beroerteonderzoek 

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

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

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

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

Medicine

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 ...

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

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

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

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 ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...

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 ...

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 ...

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 ...

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

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