Login processing...

Trial ends in Request Full Access Tell Your Colleague About Jove


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

Published: May 5, 2023 doi: 10.3791/65345


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. 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.


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.

Subscription Required. Please recommend JoVE to your librarian.


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

  1. Examine the mouse for gross abnormalities and behavioral deficits.
    NOTE: Before surgery, it's important for surgeons to wear appropriate PPE (protective personal equipment), including surgical mask, cap, gloves, and gown.
  2. Weigh the mouse; program the ventilator (see Table of Materials) based on the body weight.
  3. 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.
  4. Insert the guide wire into the 20 G intravenous (IV) catheter.
  5. Take the mouse out of the induction box when its respiratory rate is reduced to 30-40 breaths per minute.
  6. Lay the mouse on the surgical bench in a supine position. Pull the mouse's tongue out and hold it with the fingers of the left hand. Insert a laryngoscope (see Table of Materials) into the animal's mouth to visualize the vocal cord.
  7. Stabilize the mouse chin on the laryngoscope using the right middle finger. Free the left hand to hold the 20 G IV catheter.
  8. 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.
  9. 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.
    ​NOTE: Do not forget to reduce the isoflurane concentration. Otherwise, the mouse will receive an overdose of anesthesia.
  10. Apply eye ointment onto both eyes and inject 5 mg/kg carprofen subcutaneously.
  11. Keep the mouse in a lateral position with the right temporal area facing up. Maintain the rectal temperature at 37 °C using a heating pad (35 °C) and a heat lamp controlled by a temperature regulator (see Table of Materials).
  12. Shave the surface area between the right eye and ear, and disinfect the surgical area at least three times with iodine and alcohol swabs.

2. MCAO surgery

  1. 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.
  2. Dissect the underlying fascia with forceps to expose the temporal and masseter muscles.
    NOTE: Be careful not to damage the parotid gland.
  3. 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.
  4. Use the tip of a high-temperature cautery loop (see Table of Materials) to cut a 5 mm transverse incision on the temporal muscle.
  5. Use two forceps to dissect the underlying zygomatic arch and expose the joint between the maxilla and zygomatic bones.
  6. 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.
  7. 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.
  8. 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.
  9. 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.
  10. 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).
  11. For transient MCAO, tighten the silk thread knot slightly under the needle to block arterial blood flow (Figure 2E), representing MCAO onset.
  12. 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).
  13. For permanent MCAO, firmly tighten the silk thread loop around the artery, and remove the needle. Cut and remove the excess suture materials.
  14. 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.

3. Post-surgical care

  1. Turn the isoflurane off to awaken the mouse. Disconnect the ventilator when spontaneous respiration is restored.
  2. Transfer the mouse to a recovery chamber (see Table of Materials) with a controlled temperature.
  3. Extubate the mouse when its righting reflex is restored, or it begins to move.
  4. 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.

4. Laser speckle contrast imaging (LSCI)

  1. 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.
  2. Make a 3 cm midline skin incision and dissect the skin from the skull. Apply four small needle retractors to expose the skull top.
  3. Move the laser speckle camera (see Table of Materials) above the head, and adjust the focus of the camera. Image the cerebral blood flow.

5. 2,3,5-triphenyltetrazolium chloride (TTC) staining

  1. 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.
  2. Decapitate the mouse using a surgical scissor and harvest the brain. Incubate the brain in ice-cold saline for 20 min.
  3. 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.
  4. 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.
  5. Transfer the brain slices to 10% formalin for 24 h of fixation. Image the brain slices and measure the infarct area.

Subscription Required. Please recommend JoVE to your librarian.

Representative Results

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 >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 (<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.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Subscription Required. Please recommend JoVE to your librarian.


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 >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.

Subscription Required. Please recommend JoVE to your librarian.


All authors have no conflict of interest.


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).


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



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


Modified Transcranial Middle Cerebral Artery Occlusion Model Stroke Outcomes Aged Mice Experimental Stroke Research Middle Cerebral Artery Occlusion Intraluminal Filament Ischemic Stroke Cerebral Infarction Posterior Cerebral Artery Posterior Communicating Artery Atresia Mortality Rate Long-term Stroke Recovery Distal MCAO Models Cortex Area Infarction Post-stroke Neurologic Deficits Modified Transcranial MCAO Model Occlusion Location Cortex And Striatum Damage Long-term Survival Rate Neurologic Deficits Characterization
A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice
Play Video

Cite this Article

Sheng, H., Dang, L., Li, X., Yang,More

Sheng, H., Dang, L., Li, X., Yang, Z., Yang, W. A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice. J. Vis. Exp. (195), e65345, doi:10.3791/65345 (2023).

Copy Citation Download Citation Reprints and Permissions
View Video

Get cutting-edge science videos from JoVE sent straight to your inbox every month.

Waiting X
Simple Hit Counter