Table of Contents  
Year : 2011  |  Volume : 2  |  Issue : 1  |  Page : 36-39  

Protocol for middle cerebral artery occlusion by an intraluminal suture method

Department of Pharmacology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry - 605 006, India

Date of Web Publication25-Feb-2011

Correspondence Address:
M Rupadevi
Department of Pharmacology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry - 605 006
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0976-500X.77113

Rights and Permissions

How to cite this article:
Rupadevi M, Parasuraman S, Raveendran R. Protocol for middle cerebral artery occlusion by an intraluminal suture method. J Pharmacol Pharmacother 2011;2:36-9

How to cite this URL:
Rupadevi M, Parasuraman S, Raveendran R. Protocol for middle cerebral artery occlusion by an intraluminal suture method. J Pharmacol Pharmacother [serial online] 2011 [cited 2021 Sep 24];2:36-9. Available from:

   Introduction Top

Generally, the experimental animal models serve as indispensable tools to predict the value and the effect of therapeutic approaches in human subjects. A number of procedures have been followed to develop experimental models of stroke in many species. The rodents (rats and mice) are involved in the majority of neurological experiments, since they are cost effective, free of ethical burdens compared to larger animals, and especially, their cerebrovascular anatomy and physiology presents resemblance to humans. Hemorrhagic, global, and focal ischemia are the general approaches to establish the animal models of stroke. Among these approaches, the focus on the focal ischemic models has increased, with preference for middle cerebral artery occlusion (MCAo). In the MCAo procedure, there are two major variants, i.e., transient and permanent. Initially, the direct middle cerebral artery (MCA) ligation by a subtemporal approach was considered as the standard method of producing a permanent proximal MCAo but MCA occlusion by craniectomy may cause damage due to brain retraction and vessel manipulation. Apart from this disadvantage, reperfusion may not be attained easily and this technique can disturb the intracranial environment due to its surgically invasive nature. Hence, MCAo using intraluminal suture has gained increasing attention in the stroke research. MCAo by the intraluminal suture method in rodents is a widely accepted and well-standardized animal model for cerebral ischemia and reperfusion injury. Kozuimi et al. [1] introduced this model which mimics the human ischemic stroke and produces infarction in the MCA territory which involves both frontopareital cortex and lateral caudoputamen. MCAo is performed via either external carotid artery or the common carotid artery which eliminated the requirement of craniectomy and all the problems associated with an open skull procedure. The major advantage of this technique is that reperfusion can be achieved easily which helps to alter the duration of ischemia in a controlled manner. This article describes the methodology and evaluation of the MCAo model in rodents. The approval from the relevant committees (Institutional animal ethics committee) is required for all the experimental procedures.

   Preoperative Care Top

  • Healthy adult male rats weighing 200-250 g or mice weighing 25-30 g are selected for the surgery. Since estrogen is a neuroprotectant which may affect the intensity of infarction, [2] males are preferable.
  • All the animals are fasted overnight before the experiment.

   Preparation of the Monofilament for MCAo Top

Requirements include 6-0 (mice) or 4-0 (rats) nylon monofilament (Ethicon, Johnson and Johnson), a candle, forceps, and poly-L-lysine (0.1% w/v; Sigma).

  • The monofilament is cut into 2 cm (for mice) or 3 cm (for rats) pieces and the tip of the monofilament is heated for a brief period or till the tip is swollen to form a bulb shape or blunt end (all the tip ends are uniformly blunted to occlude the MCA).
  • The monofilaments are immersed in poly-L-lysine for 30 min and dried in an incubator at 37 ± 2°C for overnight. The filaments are stored for surgical purpose.
  • Poly-L-lysine is used as an adhesive subbing solution and its polycationic nature helps to facilitate a strong interaction with the anionic sites of the artery which produces a complete occlusion of the MCA.
  • The blunt end of the filament occludes the MCA properly which helps to produce ischemia in the MCA territory.
  • It is recommended to standardize the optimal tip diameter and inserted length of the suture for the animal used. To get 100% successful stroke, silicone-coated filaments are preferable, and are commercially available.

   Procedure for MCAo Top

All the surgical instruments and materials should be autoclaved and the surgical procedure should be performed under sterile conditions.

Requirements include ketamine, xylazine, normal saline, surgical gloves, adhesive tape, curved scissors, surgical blade, curved forceps, microscissors, scissors, microvascular clips, cotton thread, suture, 1-ml syringe, sterilized cotton, 2% lidocaine HCl (Xylocaine), dissection microscope (for mice), and needle (size 16 for rats).

  • Animals are anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg) intraperitoneally or induced and maintained with 3% and 1.5% isoflurane, respectively, along with 80% oxygen using a vaporizer (it has been reported that the inhalation of anesthesia is effective compared to other methods due to the better control of physiological parameters and lower mortality during 7 days postischemia). [3]
  • After turning the animal to the supine position, it is fixed to the surgical table using an adhesive tape.
  • A midline neck incision is made and the soft tissues over the trachea are retracted gently.
  • The common carotid artery (CCA) either left or right is carefully isolated from the vagus nerve and ligated temporarily using a cotton thread.
  • Generally, the CCA is bifurcated into the external carotid artery (ECA) and internal carotid artery (ICA) which flows toward the head region, and again bifurcated into the MCA and pterigopalatine artery.
  • The first bifurcation of CCA is identified, and then, the soft tissues around the ECA and ICA are gently cleared without harming the artery.
  • Then, two closely spaced permanent knots are placed at the distal part of the ECA to prevent the backflow of blood and the ECA is cut between the knots.
  • The tied section attached proximal to the CCA junction can be straightened to allow the filament to enter the ICA, and then, the second bifurcation is cleared to obtain a good view of the MCA.
  • The microvascular clip is placed in the ICA temporarily proximal to the CCA junction and the tied section of ECA is incised using the microscissors to insert the monofilament. Once the tip of the monofilament reaches the CCA junction, a knot is placed below the arteriotomy in the ECA, and then, the microvascular clip which is placed in the ICA is removed permanently to allow filament insertion.
  • The ECA stump is straightened and the filament is advanced carefully up to 11 mm, for mice, or 17-20 mm, [4] for rats, into the MCA from the CCA junction.
  • Once filament insertion into the MCA is confirmed, the microvascular clip is removed permanently from the CCA and animal's body temperature is maintained at 37 ± 2°C using a heating blanket during occlusion.
  • After the specific occlusion period, again a clip is placed in the ICA and the knot placed in the ECA stump below the arteriotomy is loosened. The intensity of the infarction greatly depends on the MCA occlusion period. The minimum 60-90 min of the occlusion period is required to obtain a reproducible infarct volume, [5] and therefore, the most common occlusion periods of the MCA are 60, 90, and 120 min for rats. [6]
  • The filament is then withdrawn carefully until the tip is near the arteriotomy.
  • After the removal of the filament, the knot is tightened in the ECA. After the confirmation of anterograde blood flow restoration (reperfusion), the midline neck incision is sewed using surgical suture.
  • To relieve pain and discomfort in the postoperative period, topical Lidocaine gel is applied on the wound and the animal receives 0.5 ml saline intraperitoneally as volume replenishment after the surgery.
  • At the end point of the study, the animals are sacrificed and the histological analysis is carried out to confirm infarction. Generally, brain infarction can be observed after 23 h of reperfusion or surgery.

   Physiological Parameters Top

  • Mean arterial blood pressure, heart rate, and blood gases can be measured to monitor the complications during surgery.
  • The measurement of the cerebral blood flow with laser Doppler flowmetry during surgery ensures the proper occlusion of the MCA.

   Procedure for Sham Surgery Top

  • For sham surgery, all the arteries are exposed for the surgical period but the filament is not inserted into the MCA. The surgical period and the anesthesia volume should be same as that for the test animal.

   Postoperative Care Top

  • Ensure that there is no subarachnoid hemorrhage, the lesion is induced and no other complications are noted.
  • The animals which are having problems during the induction of MCAo, such as excessive bleeding, prolonged operation time, and thread displaced into the pterygopalatine artery, are excluded.
  • The animal must be monitored carefully after surgery for signs of discomfort, and caged individually.

   Neurological Deficits After Stroke Induction Top

  • The acute neurological deficit of the animal is evaluated by placing the rat on the floor (normal walk = 0, inability to walk straight = 1, circling toward the paretic side = 2, fall down to the paretic side=3) and motor test (flexion of the forelimb and hind limb) after recovered from anesthesia. [7]

   Evaluation of Brain Infarction by Histological Analysis Top

Requirements include chloroform, surgical blade/brain matrix, 2, 3, 5-triphenyltetrazolium chloride (TTC), normal saline, 100-ml beaker, rat or mouse brain 10% formalin.

   Preparation of the Reagent Top

  • 0.9% NaCl
  • 0.5% TTC (for 50 ml of 0.9% NaCl, mix 500 mg of TTC).
The solution should be prepared freshly.

   Principle Top

TTC is an oxidation-reduction indicator which is used for early histochemical diagnosis of infarction and is one of the most frequently used staining method for the reliable macroscopic identification of infarction. [8],[9] The staining action of TTC is based on the oxidation of TTC by intact mitochondrial dehydrogenase, which oxidizes the tetrazolium salts into formazan, the carmine red product. Due to the absence of active dehydrogenase in the infracted or necrotic tissue, it remains unstained. Therefore, the infracted tissue can be visually identified.

   Procedure for TTC Staining Top

  • After 24 h of reperfusion, the animal is euthanized using the overdose of urethane followed by cervical dislocation and decapitated to remove the brain carefully.
  • The brain is frozen at −80°C for 15 min and sliced into 1-mm (for mice) or 2-mm (for rats) coronal sections.
  • The brain slices are immersed in the TTC solution for 15-20 min at 37°C.
  • The ischemic damage to coronal brain sections is qualified by the absence of staining and, for fixation, the brain slices are then transferred into a 10% v/v formaldehyde solution after washing with normal saline.
  • Images of brain sections are captured using a digital camera with good resolution.

   Discussion Top

Experimental ischemic stroke models render a better understanding of the mechanisms of an ischemic brain injury. Despite tremendous efforts that have been made in the last two decades, the recombinant tissue-type plasminogen activator is the only approved therapy for acute ischemic stroke within a 3-h time window, and there are many failures of neuroprotective drug clinical studies. Though many reasons can be interpreted for these failures, the drawbacks of preclinical studies such as false-positive results, inflated effect size, and marginal reproducibility have to be considered. MCAo by the intraluminal suture method is a better choice for experimental models of stroke since it may produce a more reproducible alternative for inducing stroke. Moreover, it yields highly consistent results which minimize misleading interpretations, and hence, it is considered as a reliable stroke model to test a variety of neuroprotective drugs.

   References Top

1.Koizumi J, Yoshida Y, Nakazawa T, Ooneda G. Experimental studies of ischemic brain edema: I: A new experimental model of cerebral embolism in which recirculation can introduced into the ischemic area. Jpn J Stroke 1986;8:108.  Back to cited text no. 1
2.Brann DW, Dhandapani K, Wakade C, Mahesh VB, Khan MM. Neurotrophic and neuroprotective actions of estrogen: Basic mechanisms and clinical implications. Steroids 2007;72:381-405.  Back to cited text no. 2
3.Zausinger S, Baethmann A, Schmid-Elsaesser R. Anesthetic methods in rats determine outcome after experimental focal cerebral ischemia: Mechanical ventilation is required to obtain controlled experimental conditions. Brain Res Brain Res Protoc 2002;9:112-21.  Back to cited text no. 3
4.Yousuf S, Atif F, Ahmad M, Hoda MN, Khan MB, Ishrat T, et al. Selenium plays a modulatory role against cerebral ischemia-induced neuronal damage in rat hippocampus. Brain Res 2007;1147:218-25.  Back to cited text no. 4
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;87:179-97.  Back to cited text no. 5
6.Thomas CS. Rodent models of focal stroke: Size, mechanism and purpose. NeuroRx 2005;2:396-409.  Back to cited text no. 6
7.Wakayama K, Shimamura M, Sata M, Sato N, Kawakami K, Fukuda H, et al. Quantitative measurement of neurological deficit after mild (30 min) transient middle cerebral artery occlusion in rats. Brain Res 2007;1130:181-7.  Back to cited text no. 7
8.Isayama K, Pitts LH, Nishimura MC. Evaluation of 2,3,5-triphenyltetrazolium chloride staining to delineate rat brain infarcts. Stroke 1991;22:1394-8.  Back to cited text no. 8
9.Hatfield RH, Mendelow AD, Perry RH, Alvarezs LM, Modha P. Triphenyltetrazolium chloride (TTC) as a marker for ischaemic changes in rat brain following permanent middle cerebral artery occlusion. Neuropathol Appl Neurobiol 1991;17:61-7.  Back to cited text no. 9

This article has been cited by
1 Hyperbaric Oxygen Protects Against Cerebral Damage in Permanent Middle Cerebral Artery Occlusion Rats and Inhibits Autophagy Activity
KongMiao Lu,HaiRong Wang,XiaoLi Ge,QingHua Liu,Miao Chen,Yong Shen,Xuan Liu,ShuMing Pan
Neurocritical Care. 2018;
[Pubmed] | [DOI]
2 Sox9 knockout mice have improved recovery following stroke
Xiaoyun Xu,Bethany Bass,William M. McKillop,Janina Mailloux,Tony Liu,Nicole M. Geremia,Todd Hryciw,Arthur Brown
Experimental Neurology. 2018; 303: 59
[Pubmed] | [DOI]
3 Metabolomics-based mechanisms exploration of Huang-Lian Jie-Du Decoction on cerebral ischemia via UPLC-Q-TOF/MS analysis on rat serum
Baojie Zhu,Huiting Cao,Limin Sun,Bo Li,Liwei Guo,Jinao Duan,Huaxu Zhu,Qichun Zhang
Journal of Ethnopharmacology. 2018;
[Pubmed] | [DOI]
4 Combination of Emricasan with Ponatinib Synergistically Reduces Ischemia/Reperfusion Injury in Rat Brain Through Simultaneous Prevention of Apoptosis and Necroptosis
Jing Tian,Shu Guo,Heng Chen,Jing-Jie Peng,Miao-Miao Jia,Nian-Sheng Li,Xiao-Jie Zhang,Jie Yang,Xiu-Ju Luo,Jun Peng
Translational Stroke Research. 2017;
[Pubmed] | [DOI]
5 Small chaperons and autophagy protected neurons from necrotic cell death
Ye Lei,Kai Liu,Lin Hou,Lianggong Ding,Yuhong Li,Lei Liu
Scientific Reports. 2017; 7(1)
[Pubmed] | [DOI]
6 LOTUS overexpression accelerates neuronal plasticity after focal brain ischemia in mice
Hajime Takase,Yuji Kurihara,Taka-akira Yokoyama,Nobutaka Kawahara,Kohtaro Takei,Lucio Annunziato
PLOS ONE. 2017; 12(9): e0184258
[Pubmed] | [DOI]
7 Semi-synthetic sapogenin exerts neuroprotective effects by skewing the brain ischemia reperfusion transcriptome towards inflammatory resolution
Laura García-Pupo,Jeney Ramírez Sánchez,Dariusz Ratman,Claudina Pérez-Novo,Ken Declerck,Karolien De Bosscher,Marios Nektarios Markakis,Gerrit Beemster,Armando Zaldo,Yanier Nuñez Figueredo,René Delgado-Hernández,Wim Vanden Berghe
Brain, Behavior, and Immunity. 2017;
[Pubmed] | [DOI]
8 Neuroprotective effect of lercanidipine in middle cerebral artery occlusion model of stroke in rats
Sangeetha Gupta,Uma Sharma,Naranamangalam R Jagannathan,Yogendra Kumar Gupta
Experimental Neurology. 2017; 288: 25
[Pubmed] | [DOI]
9 A Comparative Study of Variables Influencing Ischemic Injury in the Longa and Koizumi Methods of Intraluminal Filament Middle Cerebral Artery Occlusion in Mice
Gary P. Morris,Amanda L. Wright,Richard P. Tan,Amadeus Gladbach,Lars M. Ittner,Bryce Vissel,Lucio Annunziato
PLOS ONE. 2016; 11(2): e0148503
[Pubmed] | [DOI]
10 Epigallocatechin Gallate Extends the Therapeutic Window of Recombinant Tissue Plasminogen Activator Treatment in Ischemic Rats
Yi-Ping You
Journal of Stroke and Cerebrovascular Diseases. 2016;
[Pubmed] | [DOI]
11 Protective effects of mangiferin on cerebral ischemia–reperfusion injury and its mechanisms
Zhang Yang,Chen Weian,Huang Susu,Wang Hanmin
European Journal of Pharmacology. 2016; 771: 145
[Pubmed] | [DOI]
12 Activation of the Nrf2 defense pathway contributes to neuroprotective effects of phloretin on oxidative stress injury after cerebral ischemia/reperfusion in rats
Yu Liu,Lei Zhang,Jiangjiu Liang
Journal of the Neurological Sciences. 2015; 351(1-2): 88
[Pubmed] | [DOI]
13 Experimental animal models and inflammatory cellular changes in cerebral ischemic and hemorrhagic stroke
Tao Yan,Michael Chopp,Jieli Chen
Neuroscience Bulletin. 2015; 31(6): 717
[Pubmed] | [DOI]
14 The Effect of Pre-Condition Cerebella Fastigial Nucleus Electrical Stimulation within and beyond the Time Window of Thrombolytic on Ischemic Stroke in the Rats
Weiju Tang,Weiwei Dong,Peng Xie,Pengfei Cheng,Shunjie Bai,Yifei Ren,Gong Wang,Xiuying Chen,Chun Cui,Yuxiang Zhuang,Wen Huang,Honglian Shi
PLOS ONE. 2015; 10(5): e0128447
[Pubmed] | [DOI]
15 Terazosin activates Pgk1 and Hsp90 to promote stress resistance
Xinping Chen,Chunyue Zhao,Xiaolong Li,Tao Wang,Yizhou Li,Cheng Cao,Yuehe Ding,Mengqiu Dong,Lorenzo Finci,Jia-huai Wang,Xiaoyu Li,Lei Liu
Nature Chemical Biology. 2014;
[Pubmed] | [DOI]
16 Biochanin A protects against focal cerebral ischemia/reperfusion in rats via inhibition of p38-mediated inflammatory responses
Wenbo Wang,Lejian Tang,Yong Li,Yong Wang
Journal of the Neurological Sciences. 2014;
[Pubmed] | [DOI]
17 Suppression of NADPH oxidase- and mitochondrion-derived superoxide by Notoginsenoside R1 protects against cerebral ischemia–reperfusion injury through estrogen receptor-dependent activation of Akt/Nrf2 pathways
Xiangbao Meng,Min Wang,Xiaocheng Wang,Guibo Sun,Jingxue Ye,Huibo Xu,Xiaobo Sun
Free Radical Research. 2014; : 1
[Pubmed] | [DOI]
18 Long Course Hyperbaric Oxygen Stimulates Neurogenesis and Attenuates Inflammation after Ischemic Stroke
Ying-Sheng Lee,Chung-Ching Chio,Ching-Ping Chang,Liang-Chao Wang,Po-Min Chiang,Kuo-Chi Niu,Kuen-Jer Tsai
Mediators of Inflammation. 2013; 2013: 1
[Pubmed] | [DOI]
19 Influence of the time of middle cerebral artery occlusion on induction of cerebral ischemia-reperfusion injury in mice
Chen, L. and Cao, L.-X. and Qian, Y.-S. and Guan, T. and Huang, M.-H. and Huang, L.-F. and Li, Y.-M.
Chinese Journal of New Drugs. 2012; 21(11): 1292-1295+1305


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

  In this article
    Preoperative Care
    Preparation of t...
    Procedure for MCAo
    Physiological Pa...
    Procedure for Sh...
    Postoperative Care
    Neurological Def...
    Evaluation of Br...
    Preparation of t...
    Procedure for TT...

 Article Access Statistics
    PDF Downloaded1063    
    Comments [Add]    
    Cited by others 19    

Recommend this journal