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. Author manuscript; available in PMC: 2022 Jul 1.
Published in final edited form as: J Neurosci Methods. 2021 Apr 2;358:109179. doi: 10.1016/j.jneumeth.2021.109179

Modified Middle Cerebral Artery Occlusion Model Provides Detailed Intraoperative Cerebral Blood Flow Registration and Improves Neurobehavioral Evaluation

Maria Shvedova 1,2, Mohammad Rashedul Islam 1, Antonis A Armoundas 1, Nina D Anfinogenova 3, Christiane D Wrann 1,4, Dmitriy N Atochin 1
PMCID: PMC8217142  NIHMSID: NIHMS1690821  PMID: 33819558

Abstract

Background:

Middle cerebral artery occlusion (MCAO) with 1-hour ischemia followed by reperfusion is a widely used stroke model in rodents that has significant limitations such as high mortality and severe neurological deficit hampering comprehensive neurobehavioral evaluation. The goal of this study was to establish a mouse model of 30-minute MCAO followed by 48 hours of reperfusion and compare it with 1-hour MCAO followed by 24 hours of reperfusion.

New Method:

Here we propose a modified MCAO model that is favorable for both neurobehavioral and infarct volume evaluation. The model includes shorter ischemic time (30 min) of MCAO followed by 48 h of reperfusion and use of standardized intraoperative partial and total reperfusion, which allows for the detailed evaluation of initial and total reperfusion by means of the monitoring of CBF by LDF.

Results and Comparison with Existing Method:

Intraoperative CBF parameters and infarct volume (1-h MCAO at 24 hours: 69±9; 30-minute MCAO at 48 hours: 65±14 mm3) did not significantly differ between groups. Neurological deficit was less severe in 30-minute MCAO group where mice also had significantly longer ambulatory distance and time, lower resting time, and higher vertical count on the OPF. The latency to fall in the rotarod test was significantly higher in 30-minute MCAO group. The mortality was higher after 1-hour MCAO.

Conclusions:

30-minute MCAO followed by 48 hours of reperfusion causes intraoperative ischemia, reperfusion and infarct volume comparable with 1-hour MCAO followed by 24 hours of reperfusion but results in lower mortality with milder neurological deficit allowing for more extensive neurobehavioral evaluation.

Keywords: middle cerebral artery occlusion, ischemic stroke, neurological evaluation, open field test, rotarod test, murine model

1. Introduction

Deaths from stroke have decreased over the past decade, but stroke still remains a major health problem1–3. Stroke is often followed by devastating outcomes such as motor impairment, dementia, depression, and increased risk of rehospitalization, all of which contribute to the rising socioeconomic burden2. These circumstances make studies aimed at stroke treatment and prevention vital. Animal models are essential for studying new treatments of stroke.

Different modalities of middle cerebral artery occlusion (MCAO) followed by reperfusion have been used to induce permanent and transient focal cerebral ischemia in rodents4,5. MCAO is the stroke model commonly used in pre-clinical stroke studies in rats and mice since ischemic stroke in humans most frequently occurs through an occlusion of the middle cerebral artery (MCA)6. While rats are frequently utilized for stroke studies, we choose mouse MCAO model since stroke experiments often require involvement of genetically modified or molecularly manipulated mice7. Knockout and knockin mice have been used to identify the mechanisms of cell death signaling, and widely used in the pharmacological testing7. Particularly, C57Bl6 mouse strain is a common background for transgenic mouse strains development.

While 1-h MCAO model is most commonly used in pre-clinical stroke studies, it has significant limitations. The mortality rate after transient MCAO can be as high as 50% and higher in C57Bl6 mice8,9, reaching up to 71% 24 h after 1-h MCAO in Balb/C mice followed by 100% mortality 72 h after 1-h MCAO in Balb/C mice10.

High mortality rates in some mouse strains could be, at least in part, due to the high variability in cerebral vascularization in mice from different strains as was revealed by magnetic resonance angiography11. Accordingly, different mouse strains (Balb/C, BDF, and CFW) demonstrate significant differences in susceptibility to focal cerebral ischemia: Balb/C mice develop highest infarct volume after MCAO and highest mortality rate (67%), compared to no mortality in BDF mice and 42% mortality in CFW mice 24 hours after MCAO with ipsilateral common carotid artery occlusion. Ipsilateral common carotid artery occlusion alone elicits hemispheric infarctions in 36% of Balb/C mice but not in the BDF and CFW strains. Carbon black studies reveal incomplete circle of Willis (decreased occurrence of patent posterior communicating arteries) in Balb/C mice compared to BDF mice, while CFW mice demonstrate intermediate result. Authors conclude that significant difference between mouse strains in their sensitivity to cerebral ischemia is related, at least partially, to the cerebrovascular anatomy at the level of the posterior communicating arteries12.

Severe neurological deficit after 1-h MCAO compromises extensive neurobehavioral evaluation. Implementation of MCAO model with shorter time of ischemia may benefit experiments on mouse strains with high mortality or when the neurobehavioral evaluation is a primary subject of the study.

Several modalities of 30-min MCAO with varying reperfusion time have been implemented in a few studies13–15, including our two manuscripts16,17. However, this is the first study where we present and describe in detail our 30-min MCAO model, followed by 30 min of monitored CBF during standardized partial and total reperfusion and neurobehavioral evaluation after 48 hours and compare this model with the widely accepted 1-h MCAO model followed by 24-h reperfusion.

2. Materials and methods

2.1. Animals

All experiments were approved by the Massachusetts General Hospital Subcommittee on Research and Animal Care and performed in accordance with the recommendations outlined in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. We used 11–15-week-old wild type C57Bl6 mice obtained from Jackson Laboratory. Only male mice were used for this methodological study. Mice were randomly divided into two groups: group 1 comprised of the animals undergoing 1-h MCAO followed by 24 h of reperfusion (n=32); group 2 included mice undergoing 30-min MCAO followed by 48 h of reperfusion (n=20). Six additional mice for each procedure group were randomly assigned for a separate experiment with intraoperative blood pressure and arterial blood gases monitoring.

2.2. Surgical procedure of MCAO followed by reperfusion

The transient MCAO procedure was performed under general anesthesia (1.5% isoflurane in 30% O2 and 70% N2O). Both eyes were covered with an eye ointment (Puralube). A thermal probe inserted into the rectum and heating pad were used for body temperature maintenance between 36.5 °C and 37.0 °C18–20.

The general operative approach was similar to standard MCAO procedure9. Under a dissecting microscope after skin disinfection with 70% ethanol (EtOH), midline 1-cm incision was done on the skull, temporal muscles were detached, and temporal bone was cleaned with a scalpel. CBF was measured during the surgical procedure by laser Doppler flowmetry (LDF) with a fiber-optic probe attached to the skull over the core area supplied by the MCA. Then, the mouse was turned to the prone position, and, after disinfection with 70% EtOH, a 1 cm incision was performed on the midline of the neck. Salivary glands were carefully moved laterally from the midline, pretracheal muscles were pulled to the right side and sternocleidomastoid muscle was diverted to the left side. The left common carotid artery was isolated and dissected with vascular tweezers from the surrounding structures. Common carotid artery (CCA) was closed proximally and then two sterile silk ligatures (7/0) were placed on the external carotid artery (ECA) 5 and 6 mm from the bifurcation of the CCA. The superior thyroid artery was isolated and cauterized with monopolar electrocautery. The ECA was cut in between the ligatures and proximal stump of the ECA was moved counter-clockwise to position appropriate for insertion of the filament. Two ligatures were placed on the internal carotid artery 5 and 7 mm from the bifurcation of the CCA. The distal ligature was tightened on the bow node. The proximal ligature was left as a loose bow node. Small incision was performed on the distal end of the remaining proximal stump of the ECA closer to the proximal ligature. MCAO was elicited by inserting a 7-0 nylon silicon-covered filament (Doccol) into the ECA and further into the internal carotid artery (ICA) (after the advancement beyond the proximal node on the ICA, the proximal node was tightened just to allow for advancing the filament further, but preventing retrograde bleeding, and the distal node was loosened) and to the origin of the MCA. The achievement of the MCAO was confirmed via laser-Doppler monitor by the drop in CBF. The filament was fixed on place with the ligature around the ICA and left for 30 min or 1 h of ischemia. The caliber of the filament was chosen in accordance with the recommendation from the supplier’s website depending on the animals’ weight.

In our modified model, after the end of ischemia, reperfusion was performed in two distinct steps: after the withdrawal of the filament from MCA, we monitored initial reperfusion through the Willis circle for 15 min, the second step was total reperfusion which was performed after the closure of the ECA (to prevent bleeding) and achieved by the opening of the ipsilateral CCA. Total reperfusion was monitored by LDF for 15 min in each mouse. On average, total procedure time equals to 85±5 minutes in 30-min MCAO group and 115±5 minutes in 1-h MCAO group (15 minutes from the beginning of the surgery to the beginning of the MCAO, 30 or 60 minutes of the ischemia, 30 minutes of partial and total reperfusion monitoring, and 10 minutes for the wound closure). One mouse was excluded from the study due to abnormal carotid artery anatomy which resulted in the impossibility to elicit ischemia by introducing the filament into the MCA.

After suturing of the surgical wounds, mice were transferred to cages for post-surgical observation. Each mouse received 0.5 mL normal saline injection subcutaneously immediately post-surgery for fluid replacement. Mice were given buprenorphine subcutaneously (0.03 mg/kg) for analgesia every 12 h and housed with food and water at libitum; water-softened chow and recovery gel were provided in each cage. Mice were euthanized either 48 h after reperfusion (30-min MCAO model) or 24 h after reperfusion (1-h MCAO model).

2.3. Arterial blood gas monitoring

Arterial blood gas analysis and blood pressure studies were performed on a separate cohort of mice (n=6 in each group) in order to avoid the impact of additional operative stress due to the femoral artery catheterization procedure and blood loss for blood gas analysis on stroke outcome and well-being of the experimental animals. Mice were anesthetized (1.5% of isoflurane in 30% O2 and 70% N2O) and the femoral artery catheterization was performed with a PE-10 catheter that was secured on place with ligatures. The wound was covered with sterile gauze wettened in PBS, and the MCAO procedure was performed as described above. Arterial blood pressure was monitored continuously and arterial pO2, CO2, and pH were measured immediately at the end of each surgery.

2.4. Neurological deficit and neurobehavioral evaluation

2.4.1. Evaluation of neurological deficit

Neurological evaluation was performed at 24 h (1-h MCAO) and at 24 h and 48 h after reperfusion (30-min MCAO), by the following scale scoring from 0 to 4: normal motor function – 0 points; contralateral torso and forearm flexion while lifting the animal by the tail – 1 point; circling to the contralateral side – 2 points; leaning to the contralateral side at rest – 3 points; no spontaneous motor activity – 4 points16.

2.4.2. Open Field Test (OPF)

The OPF test was performed at 24 h after reperfusion in 1-h MCAO and at 48 h in 30-min MCAO. Mice were brought to the behavioral evaluation room at least 30 min prior to being placed in the OPF chamber (Med Associates) to minimize any stress from the transfer. Mice were placed in the OPF chamber for 60 min and were allowed to move freely during the trial. Each infrared beam break was recorded by the software. Between each trial, the OPF chamber was thoroughly cleaned with 70% EtOH to remove any scent of the previous animal. Mice were returned to their home cage after the 60-min trial was completed. The total distance traveled was used as a measure for locomotor activity.

2.4.3. Rotarod test

The rotarod test was performed after the OPF test was completed. Mice were placed on a rotating cylinder (Harvard Apparatus) with a rubber surface (to provide grip) at zero rotations per minute (rpm). The speed was ramped up from 4 rpm to 40 rpm over time, depending on the habituation- or trial phase. If mice could not retain proper motor coordination and grip on the rubber surface, they fall off from the rotarod. Mice fall a short distance (10 cm) onto a magnetic platform that simultaneously disrupts the magnet to signal that the test is over. Relevant information regarding each trial was recorded on a touch screen. Mice were acquainted to the rotarod test via a habituation phase consisting of two trials. During the habituation phase, the rotarod accelerated 0–40 rpm over the course of 10 min. During the following trial phases, acceleration from 0 to 40 rpm occurred over a 5-min time period. Five trials were performed for each animal. Between each trial, the rotarod rubber platform and surrounding area were cleaned with 70% EtOH. Following each trial, mice were returned to their home cages. Terminal time point was selected to perform Rotarod and open field test to avoid confounding due to habituation during repeated testing in 30-min MCAO group.

2.5. Evaluation of infarct volume

Immediately after completion of the rotarod test, mice were euthanized, and brains were removed, placed on the RBMS-200C mouse brain matrix (Kent Scientific), cut into coronal sections (2-mm-thick), and stained in the dark in PBS with 2% 2,3,5-triphenyl tetrazolium chloride for 60 minutes at 37 °C. The last slice included the cerebellum and was not analyzed because of the absence of stroke in the cerebellar region after MCAO. Infarct volume was measured with the use of the ImageJ image processing program. We calculated indirect infarct volume in each section (a non-ischemic part of the ipsilateral hemisphere) to express the area of infarction. Images were analyzed by an operator blinded to the information regarding the animal group. We excluded mice that died after surgery from the infarct volume calculations. Figure 1 represents experimental timeline.

Figure 1.

Figure 1.

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Experimental timeline. CCAL - CCA ligation, ICAL - ICA ligation, MCAO - middle cerebral artery occlusion, Partial Rf - initial reperfusion after removal of the filament, Total Rf - reperfusion through the ipsilateral CCA.

2.6. Statistical analysis

Results are expressed as mean ± SD. Distance travelled in 5 minutes time bin is presented as mean ± SEM. Distance travelled in 5 minutes time bin was analyzed using 2-way ANOVA. Fisher’s exact test was used to analyze mortality rates. Mann-Whitney U test was used to analyze neurological score. For all other experiments statistical analysis was performed with two-tailed t-test. Difference of p<0.05 was considered statistically significant.

3. Results

3.1. Cerebral blood flow

Detailed CBF measurements were performed using LDF throughout the MCAO and partial reperfusion after filament removal for 15 min followed by 15 min of total reperfusion through the ipsilateral CCA. No significant differences were found in CBF before MCAO, during the ischemia, or at any timepoint registered during the reperfusion, confirming comparable intraoperative CBF conditions in both groups (Figure 2).

Figure 2.

Figure 2.

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CBF by LDF (%) during 30-min MCAO (n=20) and 1-h (n=32) MCAO. CCAL - CCA ligation, ICAL - ICA ligation, MCAO - middle cerebral artery occlusion, Partial Rf - initial reperfusion after removal of the filament, Total Rf - reperfusion through the ipsilateral CCA. The were no significant differences in CBF during the procedure between 30-min MCAO vs 1-h MCAO model.

3.2. Infarct volume

No significant infarct volume differences between groups were found (1-h MCAO: 69±9 mm3; 30-min MCAO: 65±14 mm3) (Figure 3).

Figure 3.

Figure 3.

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A – infarct volumes (indirect ischemia) at 24 h and 48 h of reperfusion in 1-h (n=19) and 30-min (n=17) ischemia groups respectively (p – ns (not significant)). B – TTC staining of representative brain coronal sections at 24 h of reperfusion after 1-h MCAO and 48 h of reperfusion after 30-min MCAO. The bar is 5 mm-long.

3.3. Neurological deficit and neurobehavioral evaluation

3.3.1. Neurological deficit

In contrast to similar reperfusion rates and infarct volumes in 1-h and 30-min MCAO groups, the neurological deficit was significantly greater in 1-h MCAO group than in 30-min MCAO group 24 h after ischemia (2.4 points in 1-h MCAO group vs 1.7 points in 30-min MCAO group, p<0.05), but the difference became less prominent when the final timepoints between the groups were compared (2.4 points in 1-h MCAO group vs 2.1 points in 30-min MCAO group 24 h after ischemia in 1-h MCAO group and 48 h after ischemia in 30-min MCAO group, p - ns) (Figure 4). However, the neurological deficit was severe enough to make impossible to proceed with OPF and rotarod tests in 6 mice from 1-h MCAO group (five mice had neurological score of 4 and one mouse had the score of 3), while only one mouse in 30-min MCAO group was excluded from these tests due to severe neurological deficit (neurological score was 3 on the first day, but declined to 4 on the second day). In the 30-min MCAO group, there were no other animals scoring more than 3 points in neurological evaluation at the final timepoint 48 h after reperfusion.

Figure 4.

Figure 4.

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Neurological deficit in mice after 1-h (n=19) and 30-min (n=17) MCAO (*p<0.05).

3.3.2. Open field test

In OPF, total ambulatory distance, distance travelled in 5 minutes time bin (Figure 5A), ambulatory time (Figure 5B), and vertical count (Figure 5C) were significantly higher after 30-min MCAO surgery in comparison with 1-h MCAO. Mice after 1-h MCAO spent significantly more time resting in comparison with animals after 30-min MCAO (Figure 5D).

Figure 5.

Figure 5.

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Open field activities at 24 h of reperfusion after 1-h MCAO (n=13) and 48 h of reperfusion after 30-min MCAO (n=16) (*p<0.05, **p<0.001). A – distance travelled in 5 minutes time bin. B – ambulatory time in sec. C – vertical count. D – resting time in sec.

3.3.3. Rotarod test

On the rotarod, mice after 30-min MCAO had significantly longer latency to fall compared to animals after 1-h MCAO (Figure 6).

Figure 6.

Figure 6.

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Rotarod test results at 24 h of reperfusion after 1-h MCAO (n=13) and 48 h of reperfusion after 30-min MCAO (n=16) (*p<0.05).

3.4. Mortality rate

The mortality rate was higher in 1-h MCAO group (n=13; 40%) than in 30-min MCAO group (n=3; 15%), p<0.05.

3.5. Arterial blood gas monitoring

Mean arterial blood pressure, as well as arterial pH, pCO2, and pO2 were not significantly different between 1-h and 30-min MCAO surgeries (Table 1).

Table 1.

Systemic physiological parameters in MCAO surgeries

MCAO model Mean arterial pressure, onset of surgery, mm Hg Intraische-mic mean arterial pressure, mm Hg Mean arterial pressure during the reperfusion, mm Hg Postoperative blood pH Postoperative blood pCO2, mm Hg Postoperative blood pO2, mm Hg
1-h 87±5 85±4 84±2 7.32±0.07 45±16 132±26
30-min 84±4 82±9 84±6 7.35±0.03 45±3 151±10
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Mean arterial blood pressure before, during, and after MCAO; postoperative arterial pH; arterial pCO2; and arterial pO2 in 1-h (n=6) and 30-min MCAO (n=6) models (Mean±SD). P - ns.

4. Discussion

Here we described modified MCAO model that is favorable for both neurobehavioral and infarct volume (as the endpoint) evaluation. The novelty of the model is based on a shorter ischemic time of MCAO followed by 48 h of reperfusion and use of standardized intraoperative partial and total reperfusion, which allows for the detailed evaluation of partial and total reperfusion by means of the monitoring of CBF by LDF. We choose 30-min MCAO over shorter occlusion time, as shorter occlusion time is considered to be insufficient to develop reproducible stroke in mice, and 30 min is a minimal critical time for the development of reproducible ischemic lesions9. Since at 48 hours after MCAO we demonstrated significantly lower mortality rate and improved neurobehavioral outcomes, we choose 48-hour as a final time point for the 30-minute MCAO model in this study.

Mice in 30-min MCAO model had the same intraoperative ischemic and reperfusion CBF levels as mice after 1-h MCAO and developed comparable cerebral infarct volumes 48 h after reperfusion and 24 h after reperfusion, respectively, while the neurobehavioral outcome and mortality rate were significantly improved in the 30-min MCAO group. Animals after 30-min MCAO had milder neurobehavioral deficit allowing for extensive neurobehavioral testing in the postoperative period. These factors allowed us to substantially decrease the group size in 30 min MCAO group.

To exclude the influence of anesthesia duration on our results we measured arterial blood pressure, pCO2, and pO2 in a separate cohort of mice. Because these parameters were not different between mouse groups (Table 1), we concluded that anesthesia duration per se does not influence our results based on comparison between two models.

The monitoring of CBF by LDF allowed us to measure the ischemic and reperfusion values of blood flow above the core area supplied by the MCA and conclude that there were no differences in cerebral blood flow between mouse groups. These measurements provide important information since it could help to identify the CBF differences during reperfusion between studied mouse groups as we described before16. LDF not only significantly increases the accuracy of filament positioning, and therefore allows for obtaining consistent infarct volume development, but also allows for real-time monitoring of CBF during the MCAO procedure21 and reperfusion. Detailed standardized continuous monitoring of CBF may benefit pharmacological investigations and pathophysiological studies utilizing transgenic mouse models where CBF comparison between the experimental groups is an important parameter. Alternative CBF measuring techniques (MRI, OCT, laser Speckle and hydrogen clearance) are more expensive, not widely available, and are difficult to routinely implement in mouse survival stroke experiments aimed at the infarct volume assessment. In our experience, LDF proved to be a reliable tool that allowed us to register the induction of MCAO, the stability of ischemia, and levels of reperfusion. The modification of the MCAO procedure which we propose here involves standardized CBF measurement during the reperfusion (15 minutes of CBF through the Willis circle after filament withdrawal, and 15 minutes of reperfusion through the ipsilateral common carotid artery). We found this standardized monitored reperfusion to be useful for our studies since it allows for detailed comparative reperfusion evaluation.

It is well known that ischemic stroke continuously develops after ischemia and its volume increases over time22. For example, the lesions in the amygdala are more developed 24 h after reperfusion than after 4 h9. Infarct volume expansion after ischemia is a possible explanation of the registered similar infarct volumes between 30-min and 1-h MCAO models, since the later final timepoint of 48 h after 30-min ischemia could allow for the development of greater ischemic lesion volume. A zone of necrosis in ischemic stroke is surrounded by the ischemic penumbra area, which is a functionally silent tissue resulting from partial blood flow deficiency23. This area remains metabolically active and represents the target region for therapy aimed at salvage of the brain tissue remaining alive after stroke24. Neurons in the penumbra region may undergo apoptosis within several hours and days after ischemia25,26. Keeping animals for 48 h after 1-h MCAO might allow for even greater infarct volume development, but since the mortality rate is high even in wild-type mice, this type of experimental design might not be suitable for many experimental goals. Further experiments aimed at the penumbra detection after MCAO in male and female mice will broaden our understanding of the observed results.

It was also described that the longer occlusion time increases the likelihood of ischemic injury in the thalamus, hippocampus, and amygdala9, which could contribute to the development of more severe neurological deficit after 1 h of ischemia in comparison with 30-min ischemia. Accordingly, it was demonstrated that hippocampal neuronal CA1 damage and mortality over 7 days in Balb/C mice after MCAO are related to the duration of brain ischemia12. Therefore, decreasing ischemic time may alleviate mortality burden in mouse stroke studies, even in such mouse strains as Balb/C, which are inherently prone to develop high mortality rate after stroke experiments due to their cerebrovascular anatomical features12.

Inflammatory response is a well-known factor contributing to infarct volume expansion after ischemic stroke, stroke development and remodeling27,28. Ischemic stroke elicits matrix metallo-proteinase-9 upregulation, increasing permeability of the blood-brain barrier29. Peripheral immune cells, such as T lymphocytes, migrate to the brain and contribute to the infarct development, increasing stroke volume30. Stroke continues to develop over time, and after transient MCAO in mouse brain tissue, activated microglia/macrophages are increased 18 h post occlusion, peaking at 48 h and remaining abundant 6 days post occlusion. Neutrophils are significantly increased by 48 h, and remain elevated 7 days post occlusion, while T lymphocytes are increased later at 72 and 96 h after occlusion staying elevated 7 days post occlusion31–33. Leukocyte infiltration presents in perfusion deficient areas in patients with acute hemispheric ischemic stroke, persisting for no less than 5 weeks after onset and then declines34. Development of inflammatory response contributes to infarct volume expansion and makes stroke models with longer post stroke survival time more suitable for studies of the inflammatory changes and therapeutic approaches aimed at the immune system modulation.

It was also shown that temporal MCAO results in skeletal muscle atrophy,35 and future studies could elucidate possible roles of the skeletal muscle abnormalities in better neurobehavioral performance in our model as compared with the 1-h MCAO model.

MCAO experiments in C57Bl6 wild type mice are often accompanied by a substantial mortality rate9. Some transgenic mouse strains are difficult to breed and perform in vivo physiological experiments on, therefore decreasing group size via decreasing mortality rate and improving animals’ ability to undergo neurobehavioral evaluation is an important area of investigation. Here we provide a modified MCAO model for preclinical stroke studies, demonstrating decreased mortality and improved neurobehavioral outcome allowing us to perform the extensive neurobehavioral evaluation in comparison with 1-hour MCAO. Due to these characteristics, described 30-min MCAO model may substantially decrease the size of the experimental animal group, which may benefit the experiments utilizing transgenic mice.

In conclusion, the 30-min MCAO followed by 48 h of reperfusion has a great potential for use in experiments aimed at detailed neurobehavioral evaluation after stroke, additionally our model provides detailed intraoperative ischemia evaluation. Decreased mortality rate after 30-min MCAO might benefit mouse stroke experiments with prohibitively high postoperative mortality.

Highlights:

  • Modified MCAO model provides detailed intraoperative reperfusion monitoring.

  • 30-min MCAO followed by 48 h of reperfusion decreases mortality compared to 1-h MCAO.

  • 30-min MCAO followed by 48 h of reperfusion improves neurobehavioral evaluation.

Funding

This work was supported by grants from the NIH [NINDS R01 NS-096237 to Dmitriy N. Atochin; R00NS087096-05 to Christiane D. Wrann; 1 R01 HL135335-01, 1 R21 HL137870-01 and 1 R21EB026164-01 to Antonis A. Armoundas].

Footnotes

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Disclosure/Conflict of Interest

None.

References

  • 1.Meschia JF, Bushnell C, Boden-Albala B, et al. Guidelines for the primary prevention of stroke: A statement for healthcare professionals from the American heart association/American stroke association. Stroke 2014; 45: 3754–3832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Joo H, Wang G, Yee SL, et al. Economic Burden of Informal Caregiving Associated With History of Stroke and Falls Among Older Adults in the U.S. Am J Prev Med 2017; 53: S197–S204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Levine DA, Wadley VG, Langa KM, et al. Risk factors for poststroke cognitive decline: The REGARDS study (reasons for geographic and racial differences in stroke). Stroke 2018; 49: 987–994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Carmichael ST. Rodent models of focal stroke: Size, mechanism, and purpose. NeuroRx 2005; 2: 396–409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Durukan A, Tatlisumak T. Acute ischemic stroke: Overview of major experimental rodent models, pathophysiology, and therapy of focal cerebral ischemia. Pharmacol Biochem Behav 2007; 87: 179–197. [DOI] [PubMed] [Google Scholar]
  • 6.McCabe C, Arroja MM, Reid E, et al. Animal models of ischaemic stroke and characterisation of the ischaemic penumbra. Neuropharmacology 2018; 134: 169–177. [DOI] [PubMed] [Google Scholar]
  • 7.Lee S, Hong Y, Park S, et al. Comparison of surgical methods of transient middle cerebral artery occlusion between rats and mice. J Vet Med Sci 2014; 76: 1555–1561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Wang R, Zhao H, Li J, et al. Erythropoietin attenuates axonal injury after middle cerebral artery occlusion in mice. Neurol Res 2017; 39: 545–551. [DOI] [PubMed] [Google Scholar]
  • 9.Morris GP, Wright AL, Tan RP, et al. A comparative study of variables influencing ischemic injury in the longa and koizumi methods of intraluminal filament middle cerebral artery occlusion in mice. PLoS One 2016; 11(2): e0148503. DOI: 10.1371/journal.pone.0148503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Schulte-Herbrüggen O, Klehmet J, Quarcoo D, et al. Mouse Strains Differ in Their Susceptibility to Poststroke Infections. Neuroimmunomodulation 2006; 13: 13–18. [DOI] [PubMed] [Google Scholar]
  • 11.Beckmann N High resolution magnetic resonance angiography non-invasively reveals mouse strain differences in the cerebrovascular anatomy in vivo. Magn Reson Med 2000; 44: 252–258. [DOI] [PubMed] [Google Scholar]
  • 12.Barone FC, Knudsen DJ, Nelson AH, et al. Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy. J Cereb Blood Flow Metab 1993; 13: 683–692. [DOI] [PubMed] [Google Scholar]
  • 13.Wiesmann M, Zinnhardt B, Reinhardt D, et al. A specific dietary intervention to restore brain structure and function after ischemic stroke. Theranostics 2017; 7: 493–512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Trueman RC, Diaz C, Farr TD, et al. Systematic and detailed analysis of behavioural tests in the rat middle cerebral artery occlusion model of stroke: Tests for long-term assessment. J Cereb Blood Flow Metab 2017; 37: 1349–1361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Schwengel K, Namsolleck P, Lucht K, et al. Angiotensin AT2-receptor stimulation improves survival and neurological outcome after experimental stroke in mice. J Mol Med 2016; 94: 957–966. [DOI] [PubMed] [Google Scholar]
  • 16.Shvedova M, Litvak MM, Roberts JD, et al. cGMP-dependent protein kinase I in vascular smooth muscle cells improves ischemic stroke outcome in mice. J Cereb Blood Flow Metab 2019; 39: 2379–2391. DOI: 10.1177/0271678X19870583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Atochin DN, Schepetkin IA, Khlebnikov AI, et al. A novel dual NO-donating oxime and c-Jun N-terminal kinase inhibitor protects against cerebral ischemia-reperfusion injury in mice. Neurosci Lett 2016; 618: 45–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Percie du Sert N, Alfieri A, Allan SM, et al. The IMPROVE Guidelines (Ischaemia Models: Procedural Refinements Of in Vivo Experiments). J Cereb Blood Flow Metab 2017; 37: 3488–3517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Belayev L, Khoutorova L, Atkins K, et al. LAU-0901, a novel platelet-activating factor antagonist, is highly neuroprotective in cerebral ischemia. Exp Neurol 2008; 214: 253–258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Chaparro RE, Izutsu M, Sasaki T, et al. Sustained functional improvement by hepatocyte growth factor-like small molecule BB3 after focal cerebral ischemia in rats and mice. J Cereb Blood Flow Metab 2015; 35: 1044–1053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Ansari S, Azari H, McConnell DJ, et al. Intraluminal middle cerebral artery occlusion (MCAO) model for ischemic stroke with laser doppler flowmetry guidance in mice. J Vis Exp 2011; 2879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Liu F, Schafer DP, McCullough LD. TTC, Fluoro-Jade B and NeuN staining confirm evolving phases of infarction induced by middle cerebral artery occlusion. J Neurosci Methods 2009; 179: 1–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Manning NW, Campbell BC V, Oxley TJ, et al. Acute ischemic stroke: time, penumbra, and reperfusion. Stroke 2014; 45: 640–4. [DOI] [PubMed] [Google Scholar]
  • 24.Hakim AM. Ischemic penumbra: The therapeutic window. Neurology 1998; 51(3 Suppl 3): S44–46. DOI: 10.1212/wnl.51.3_suppl_3.s44. [DOI] [PubMed] [Google Scholar]
  • 25.Broughton BRS, Reutens DC, Sobey CG. Apoptotic mechanisms after cerebral ischemia. Stroke 2009; 40: e331–9. DOI: 10.1161/STROKEAHA.108.531632. [DOI] [PubMed] [Google Scholar]
  • 26.Van Hemelrijck A, Hachimi-Idrissi S, Sarre S, et al. Post-ischaemic mild hypothermia inhibits apoptosis in the penumbral region by reducing neuronal nitric oxide synthase activity and thereby preventing endothelin-1-induced hydroxyl radical formation. Eur J Neurosci 2005; 22: 1327–1337. [DOI] [PubMed] [Google Scholar]
  • 27.Amantea D, Micieli G, Tassorelli C, et al. Rational modulation of the innate immune system for neuroprotection in ischemic stroke. Frontiers in Neuroscience 2015; 9: 147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Im JH, Yeo IJ, Park PH, et al. Deletion of Chitinase-3-like 1 accelerates stroke development through enhancement of Neuroinflammation by STAT6-dependent M2 microglial inactivation in Chitinase-3-like 1 knockout mice. Exp Neurol 2020; 323: 113082. [DOI] [PubMed] [Google Scholar]
  • 29.Cheng Z, Wang L, Qu M, et al. Mesenchymal stem cells attenuate blood-brain barrier leakage after cerebral ischemia in mice. J Neuroinflammation 2018; 15(1): 135. DOI: 10.1186/s12974-018-1153-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Dziennis S, Mader S, Akiyoshi K, et al. Therapy with recombinant T-cell receptor ligand reduces infarct size and infiltrating inflammatory cells in brain after middle cerebral artery occlusion in mice. Metab Brain Dis 2011; 26: 123–133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Gelderblom M, Leypoldt F, Steinbach K, et al. Temporal and spatial dynamics of cerebral immune cell accumulation in stroke. Stroke 2009; 40: 1849–57. [DOI] [PubMed] [Google Scholar]
  • 32.Stevens SL, Bao J, Hollis J, et al. The use of flow cytometry to evaluate temporal changes in inflammatory cells following focal cerebral ischemia in mice. Brain Res 2002; 932: 110–119. [DOI] [PubMed] [Google Scholar]
  • 33.Vogelgesang A, Becker KJ, Dressel A. Immunological consequences of ischemic stroke. Acta Neurologica Scandinavica 2014; 129: 1–12. [DOI] [PubMed] [Google Scholar]
  • 34.Wang PY, Kao CH, Mui MY, et al. Leukocyte infiltration in acute hemispheric ischemic stroke. Stroke 1993; 24: 236–40. [DOI] [PubMed] [Google Scholar]
  • 35.Desgeorges MM, Devillard X, Toutain J, et al. Pharmacological inhibition of myostatin improves skeletal muscle mass and function in a mouse model of stroke. Sci Rep 2017; 7: 14000. DOI: 10.1038/s41598-017-13912-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

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