Investigating Ischemic Stroke Recurrent Injury in a Secondary Middle Cerebral Artery Occlusion Model in Mouse

Summary

Here, we present a method for secondary middle cerebral artery occlusion in mice to investigate the progression of recurrent ischemic stroke. The surgical procedures, postoperative care, neuronal pathological alterations, and survival rate are described.

Abstract

Stroke is the second leading global cause of mortality and disability, with recurrent strokes contributing significantly to poor outcomes and increased neurological burden. Despite advances in acute clinical stroke management, experimental models for recurrent ischemic stroke remain limited due to their procedural complexity and high mortality rates. In this study, we aimed to establish a recurrent ischemic stroke in a mouse model and evaluate the brain pathological changes and functional deficits using a two-stage middle cerebral artery occlusion (MCAO) protocol. Adult male C57BL/6J mice underwent transient MCAO under Laser Doppler blood flow monitoring. A secondary MCAO was performed 14 days after the initial surgery to simulate stroke recurrence. Postoperative assessments included locomotor activity analysis, neurobehavioral deficits scoring by using a six-point scoring system, inflammatory markers including hypoxia-inducible factor-1α (HIF-1α) and tumor necrosis factor-α (TNF-α), and histopathological evaluation using hematoxylin and eosin (H&E) staining. After recurrent MCAO administration, mice exhibited significantly reduced locomotor activity, decreased movement speed, and diminished neurobehavioral scores, as well as increased inflammatory markers, compared to control groups. Histopathological detection revealed moderate to severe neuronal cell necrosis, neuropil vacuolation, and localized hemorrhage in the cortex and hippocampus of recurrent MCAO mice. The inflammatory markers were also induced in this recurrent MCAO procedure. This two-stage MCAO mouse model presents, for the first time, a method that effectively simulates recurrent ischemic stroke with consistent neurological and pathological outcomes and improves postoperative survival. This model could provide a reliable platform for mechanistic studies and may be applied in the future evaluation of neuroprotective therapies for recurrent stroke.

Introduction

Stroke remains a leading cause of death and long-term disability worldwide, posing a significant clinical and socioeconomic burden. More than 30 million people worldwide are affected by stroke and its consequences, including mobility loss, cognitive impairment, and the increased risk of vascular diseases^1,2. Despite advances in acute treatment such as thrombolysis and endovascular thrombectomy, many survivors remain at high risk of stroke recurrence, which is associated with worsened prognosis, greater disability, and increased mortality rates. Recurrent strokes occur in approximately 10-20% of patients within 90 days of a primary event, with the highest risk during a period of transient ischemic attack or minor stroke³. More importantly, recurrent stroke often affects vascular territories and results in more extensive infarction and severe injury, such as brain hemodynamic impairment⁴, acute and chronic immune dysregulation, and subcortical neuronal necrosis⁵, suggesting that the brain may become more vulnerable and cause more complications following an initial ischemic insult.

Some recent studies have performed ischemic or hemorrhagic stroke models in experimental animals and have revealed region-specific injury patterns, disrupted neurovascular responses, and different endogenous repair mechanisms to fit human clinical conditions^6,7. However, the research models of recurrent stroke remain less established because of their complicated procedures and high mortality rate. Some previous studies have established a photothrombotic mouse model, which emerged as a valuable tool for studying recurrent ischemic stroke^8,9,10. They demonstrated the exacerbated neuronal injury, prolonged inflammatory response, and cognitive decline in mice with recurrent stroke, and explored the underlying mechanisms, including multi-infarct dementia and inflammatory priming^8,9,10.

Another study has shown a novel aged mouse model of recurrent intracerebral hemorrhage in the bilateral striatum, which was injected with collagenase twice, resulting in delayed recovery in locomotor function, and caused cognitive loss and neurological injury in the recurrent mice¹¹. It is worth noting that the photothrombotic and hemorrhagic stroke mouse models frequently induce lesions in distinct anatomical regions during recurrent stroke, reflecting inherent methodological differences. Such variations generate heterogeneous patterns of neuronal injury, disrupt microcirculatory dynamics, and exert divergent effects on peripheral immune responses^11,12. By contrast, the MCAO model, widely regarded as the most representative rodent paradigm of ischemic stroke, offers the advantage of procedural stability and permits precise control over both the site and duration of cerebral occlusion. Nevertheless, MCAO and recurrent MCAO have also demonstrated a substantially high risk of mortality^13,14, in which imposes significant limitations on their broader applicability in experimental research. In this study, we aimed to establish a stable and reproducible two-stage MCAO mouse model that mimics recurrent stroke. We evaluated the associated neuronal injury, functional impairments, and histopathological changes. This model also demonstrated a stable survival rate, suggesting its applicability for future research on recurrent stroke pathophysiology and neuroprotective therapeutic strategies.

Protocol

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University (Study Plan ID20230335) and were conducted following established ethical guidelines for the care and use of laboratory animals.

1. Animal preparation

1. House 8-week-old male C57BL/6J mice weighing 20-25 g under controlled environmental conditions at 22 ± 1 °C with 55% relative humidity and a 12 h light/12 h dark cycle. Provide humane care with free access to food and distilled water.
2. Randomly assign 28 male mice into three groups: sham (n = 8), MCAO (n = 10), and recurrent MCAO (n = 10). Quarantine animals for 2 weeks (−d14); perform acclimatization, weighing, and grouping at −d7; administer MCAO at d0; conduct behavioral testing at d0, d2, d14, and d16; and perform recurrent MCAO surgery at d14.

2. First surgical MCAO administration

1. Sterilize all surgical instruments by autoclaving, weigh each mouse, administer carprofen orally at 5 mg/kg 1 h before the initial incision, and induce anesthesia with 3% isoflurane in an oxygen-mixed gas.
2. Monitor respiration rate (100-120 breaths/min, BPM) and evaluate the depth of anesthesia by checking the eyelid and the swallowing reflex. Position the subject on an electronic heating plate to sustain body temperature at 37 °C.
3. Shave the back and neck near the head and remove the fur and debris of mice using a vacuum. Apply ophthalmic ointment, scrub the skin, and disinfect by aseptic procedures for rodent survival surgery.
4. Use scissors and skin forceps to make the initial 0.5 cm incision between the ears to expose the left side of the central fusion line and posterior to the bregma on the skull. Remove the soft tissue on top of the skull using forceps.
5. Center the Laser Doppler adaptor in the brain of the mouse and affix it with tissue glue at a position 3 mm lateral to the left and 2 mm caudal to the bregma, ensuring the adaptor edge is pretrimmed to fit the skull base, then secure the Laser Doppler cap with adhesive to enable continuous blood flow monitoring.
   NOTE: In this step, affix the Laser Doppler cap securely at the bregma using tissue glue, positioning it at the intersection of the coronal and sagittal sutures on the superior midline of the calvaria to ensure stable monitoring of cerebral blood flow.
6. When confident with the sectioned area, draw out tissue glue using the micropipette and apply the glue around the base of the adaptor.
7. Prepare dental cement and apply a sufficient amount to surround the Doppler cap to ensure its stability throughout the procedure.
8. Rotate the animal to the supine position and make a 1 cm skin incision from the thoracic inlet towards the chin along the midline of the neck.
9. Continue with blunt dissection to separate the posterior belly of the digastric, sternohyoid, and sternomastoid muscles, followed by the use of retractors to gently pull the muscles away to expose the common (CCA), external (ECA), and internal carotid arteries (ICA), and place a section of 6-0 silk around the CCA.
   NOTE: In this step, the salivary glands should be turned upwards to facilitate the isolation of the bifurcation of the CCA and ECA. The ICA is located superior to the CCA. Blunt dissection with skin forceps is used to prevent vascular rupture and isolate the surrounding soft tissue and nerves.
10. Locate a small branch (superior thyroid artery, STA) protruding from the ECA. Use the bipolar cauterizer to burn this small branch. Proceed to cut through the burnt vessel; this will further isolate the ECA.
    NOTE: STA is typically the first branch of the ECA emerging below the level of the greater horn of the hyoid bone. To prevent injury, use a bipolar cauterizer to burn the vessel branch to achieve hemostasis.
11. Place two sections of 6-0 Silk suture around ECA. Make a permanent knot at the distal side of the ECA and a loose knot at the bifurcation site.
12. Place the Laser Doppler needle probe into the probe adaptor on the skull. Ensure that the acquisition software shows a cerebral blood flow (CBF) reading value. Record the baseline reading.
    NOTE: The standardized measure of CBF obtained by Laser Doppler was presented as perfusion units (PU), with baseline values typically calibrated within the range of 350 to 450. If reduced perfusion unit (PU) values are observed, adjust the position of the Laser Doppler probe adaptor in the mouse to ensure accurate measurement (see step 2.5).
13. Clamp the ICA using the microvascular vessel clamp and use the 6-0 silk section around the CCA to make a slip knot to ligate the CCA temporarily.
    NOTE: In this step, a reduction of CBF is detected on the Laser Doppler monitor.
14. Prepare the silicone rubber-coated nylon monofilament. Mark the filament using the silver Sharpie at 9-10 mm from the tip of the silicone head, and use microsurgical scissors to make a small incision between the tight suture and the loose suture on the ECA.
    NOTE: The residual blood in the incision site should be cleaned and rinsed with phosphate-buffered saline (PBS) to preserve vascular integrity.
15. Carefully insert the filament (silicone head first) into the ECA via the small incision.
    NOTE: This filament insertion step is omitted in the sham animals.
16. When about half of the silicone head has been inserted, slightly tighten the loose suture around the artery and the filament, and remove the clamp on the ICA.
17. Cut through the ECA underneath the first tight knot, carefully rotate the ECA vessel and filament down (counterclockwise) so that the silicone head points upwards, and push the tip of the filament up towards the ICA.
    NOTE: This step is critical and must be performed slowly in a counterclockwise direction to minimize vascular disruption and reduce the risk of entanglement or rupture.
18. Once the whole silicone part is in the blood vessel, slightly tighten the holding suture, loosen the slip knot on the CCA, and continue to push the filament in, using the silver marking on the filament for position guidance.
19. Continuously monitor the Laser Doppler software, and when resistance prevents further advancement of the filament under appropriate force while a sharp decline in CBF is observed, terminate insertion into the distal middle cerebral artery.
    NOTE: The reduction of CBF to less than 30% of the preischemic condition is a standard procedure known as Longa's method¹⁵.
20. Keep the filament within MCA for 40 min to induce brain ischemic injury.
21. Remove the filament after the designated 40 min ischemic period to reestablish CBF, apply tissue glue to achieve hemostasis, record the corresponding restored CBF measurement, detach all retractors, and remove the skull-mounted adaptor.
22. Close the skin incision near the vessel with a wound clip, maintain continuous monitoring of body temperature at 37 °C, and evaluate the respiratory rate. After confirming stable blood pressure and respiration within 160-200 BPM, discontinue anesthesia and allow recovery.

3. Second MCAO administration

1. Remove wound clips 14 days after the initial MCAO surgery, then repeat steps 2.1 through 2.9 to accomplish skull preparation, secure Laser Doppler probe placement, and expose the CCA.
2. Isolate CCA from surrounding tissue and place three segments of 6-0 silk suture around CCA.
3. Place the Laser Doppler probe in the adaptor and record the baseline CBF.
4. Make a slip knot at the distal side of CCA, a permanent knot at the proximal end, and a loose knot in between.
5. Prepare the silicone rubber-coated nylon of the occlusion filament for MCAO administration according to the described Step 2.14.
6. Make a small incision on the CCA close to the permanent and insert the filament into the CCA.
   NOTE: The residual blood in the incision site should also be cleaned and rinsed with PBS to preserve vascular integrity. This is very important.
7. Slightly tighten the loose knot and release the slip knot. Advance the filament into the ICA until the filament is under appropriate force and a marked reduction in CBF is detected.
   NOTE: The reduction of CBF to less than 30% of the preischemic condition is also considered a standard procedure.
8. Maintain the filament in position for 40 min to induce ischemic injury, as specified in step 2.20.
9. Upon completion of the designated ischemic interval, withdraw the filament and close the skin incision following the procedures outlined in steps 2.21 and 2.22 of the initial MCAO surgery.

4. Post surgery care

1. Administer warm normal saline subcutaneously at 10 mL·kg^-1·day^-1 twice daily for 3 consecutive days to prevent dehydration following MCAO surgery. Apply 10% povidone-iodine solution to the surgical site and administer amoxicillin (10 mg/kg) to prevent infection. Orally provide carprofen (5 mg/kg) for 3 days to alleviate postoperative stress and facilitate recovery under veterinary supervision after both the first and second MCAO procedures.
2. Conduct daily assessments of the health status of mice. Record changes in appearance, as well as food and water consumption during the first and second procedures.

5. Animal locomotor activity and neurobiological injury assessment

1. Evaluate locomotor activity by assessing movement trajectory, distance, and velocity. Position mice within test boxes (10 x 20 x 15 cm) and configure under a low luminosity condition.
2. Acclimate the mouse to the testing environment through a 30 min stabilization period, followed by a 10 min formal assessment. Record and analyze all parameters, including distance moved (cm), velocity (cm/s), movement duration (s), in-zone total duration (s), latency of first occurrence (s), and heading degrees (times) to determine locomotor activity.
3. Assess neurobiological injury using a modified motor function scoring system (0-6 points)¹⁶ and referencing mNSS scores (in part of motor function assessment, 6 points)¹⁷.
4. Define injury levels as follows: score 0 for absence of neurological deficit, score 1 for forelimb flexion affecting the wrist or shoulder, score 2 for forelimb flexion accompanied by reduced resistance to lateral push, score 3 for unidirectional circling toward the deficit side, score 4 for longitudinal spinning toward the deficit side, score 5 for longitudinal spinning accompanied by seizure activity, and score 6 for complete loss of movement.

6. Hematoxylin and eosin staining protocol

1. Fix MCAO and recurrent MCAO brain tissue in 10% formaldehyde buffer, and dissect the specimen into four equal parts using a 1 mm coronal mouse brain slicer. Process the third anterior portion for pathological evaluation.
2. Perform rehydration of paraffin-embedded samples: xylene for 10 min, 95% ethanol for 10 min, 75% ethanol for 10 min, 50% ethanol for 10 min, distilled water (ddH₂O) for 10 min, and PBS for 10 min.
3. Stain the samples: hematoxylin stain for 45 s and rinsing in warm ddH₂O at 30 °C for 1 min; eosin stain for 30 s and in cold ddH2O for 45 s.
4. Dehydrate and dry the samples in 75%, 95%, 100% ethanol and in xylene for 45 s each, and air dry for 60 s.
5. Mount the slides and store them in a dark chamber.
6. Assess lesion severity according to an established criterion on a five-point scale: 1 = minimal (<1%); 2 = slight (1-25%); 3 = moderate (26-50%); 4 = moderate to severe (51-75%); 5 = severe (76-100%). Conduct a pathological examination of brain tissue under blinded evaluation by a veterinary pathologist.

7. Western blotting protocol

1. Homogenize brain tissues from MCAO and recurrent mice in RIPA buffer supplemented with protease and phosphatase inhibitor (sodium fluoride 30 mM, sodium orthovanadate 30 mM, and sodium pyrophosphate 30 mM).
2. Centrifuge lysates at 10,000 x g and collect the supernatant. Quantify the supernatant protein concentration using a bicinchoninic acid (BCA) assay.
3. Perform western blotting as previously described¹⁸: load 20 µg of denatured total protein, prepared by boiling in 100 °C boiling water, into 8-12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE).
4. Transfer with 100 V for 90 min to separate proteins onto PVDF membranes in the cold room and block membranes with 3% bovine serum albumin for 60 min.
5. Incubate overnight at 4 °C with tumor necrosis factor alpha (TNF-α, 1:1000 dilutions), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:2,000 dilutions), and hypoxia-inducible factor 1 alpha (HIF-1α, 1:1,000 dilutions). Incubate membranes with appropriate secondary antibodies for 1 h.
6. Detect protein expression using an enhanced chemiluminescence substrate kit (ECL) and visualize with a digital photo-imaging system.

8. Statistical analysis

1. Present data as mean ± standard error of the mean (SEM) and perform statistical analysis using one-way analysis of variance (ANOVA), followed by Tukey's post hoc test to determine differences among multiple groups. Interpret results as statistically significant when p < 0.05.

Results

Locomotor activity and neuronal injury assessment in first and recurrent MCAO mice
In the first MCAO procedures, a silicone rubber-coated nylon monofilament was inserted from the ECA, ICA to MCA for 40 min. The cerebral blood flow (CBF) could be detected to drop conspicuously (drops <30% of baseline) by the Laser Doppler equipment, when the filament was staying in the MCA area. In this procedure, all mice stably achieved this threshold and survived. Some previous studies have indicated that the bo...

Discussion

Stroke remains the second leading cause of mortality and disability worldwide, with a high risk of recurrent stroke. Currently, despite advances in the development of medical technology and strategies, the prevention of stroke and its recurrence remains insufficient for improvement. In recent decades, various study models of stroke have been widely used in the research investigation of ischemic stroke injury and mechanisms. Among them, the MCAO surgical method is considered to be the closest to human ischemic stroke diso...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-0 silk suture	Shin-Teh Inc. TW.	ST1N-ST6
1-mm coronal mouse brain slicer	Thermo Fisher Scientific Inc., USA	V11335
Animal hair clipper	URBANER. Inc. TW	MB033
Coronal mouse brain slicer	Zivic instrument. Inc USA	BSMAS001-1
Digital photo-image system	Azure Biosystem, Inc., Dublin, CA, USA	Azure -300
Electronic bipolar cauterizer	Indiamart	AARON 940
Electronic heat plate	Reptizoo	AHM23
Eye ointment	Lodi Veterinary Care. US	17033-21-38
Laser Doppler detector	Moor instruments UK	moorVMS-LDF1-HP
Laser Doppler probe adaptor	Thermo Fisher Scientific Inc.	05-700-76
Locomotor activity detector	Orchid scientific Inc.	All Maze tracking software.
(All Maze System)	Orchid scientific Inc.	Trackezi. ver 7.39
Microvascular vessel clamp	Shin-Teh Inc. TW.	ST-M104
Microsurgical vessel scissors	Shin-Teh Inc. TW.	ST-V108
Respiration rate monitor	SDR scientific Ltd. US	SAR-830/AP
Silicone rubber-coated nylon monofilament	Fisher scientific	NC1773962
Skin forceps	Shin-Teh Inc. TW.	ST-H212
Skin scissors	Shin-Teh Inc. TW.	ST-S014PK
Sterile Cotton swab	Winner Medical	401 003
Transfer system	Semi-Dry system. Bio-rad Laboratory, USA)	1703940
Chemicals
10% povidone–iodine solution	Yoshida Pharmaceutical Co., Ltd JAP	PVP–I 10%
75% Ethanol solution	Sigma-Aldrich. USA	111727
Amoxicillin	Sigma-Aldrich, USA	A5955
Bovine serum albumin	Sigma-Aldrich, USA.	A2153
Carprofen	Plexx Inc. UK	MD150-2
GAPDH antibody	Santa Cruz Biotechnology, CA, USA	Sc-32233
H&E solution	BioPioneer Tech. CO. TW	G1005
Hypoxia-inducible factor 1 alpha antibody	Cell Signaling Technology, MA, USA	#36169
Isoflurane (anesthetic)	Life Science	R510-22-10
Sterile normal saline	Nova-Tech Inc. USA	1760RX
tumor necrosis factor alpha	Cell Signaling Technology, MA, USA	#3707