Handcrafted Silicone Coated Filament for Mice Middle Cerebral Artery Occlusion Models
Summary
This protocol describes a straightforward method for creating coated filaments for the middle cerebral artery occlusion (MCAO) model in mice using silicone, nylon sutures, and syringe needles. This method allows for the production of filaments with a consistent diameter and various silicone wrapping lengths tailored to experimental needs.
Abstract
As the global population ages, ischemic stroke has risen to become the second leading cause of disability and mortality worldwide, placing an immense burden on both society and families. Although treatments such as intravenous thrombolysis and endovascular interventions can substantially improve the outcomes for patients with acute ischemic stroke, only a small percentage of individuals benefit from these therapies. To advance our understanding of the disease and to discover more effective treatments, researchers are continuously developing and refining animal models. Among these, the middle cerebral artery occlusion (MCAO) model stands out as the most commonly used model in cerebrovascular disease research. The filament used in this model is crucial for its development. This protocol outlines a method for creating filaments with consistent diameters and varying lengths of silicone coating. The MCAO model produced using this method in C57 mice has demonstrated high success and consistency, offering a valuable tool for tailored investigations into ischemic cerebrovascular diseases.
Introduction
Stroke is one of the most prevalent causes of mortality and disability worldwide. Ischemic and hemorrhagic strokes are the primary types of cerebrovascular event, with ischemic strokes accounting for approximately 87% of cases1,2,3. Currently, there are two treatment modalities for patients with ischemic stroke: pharmacological therapy with recombinant tissue plasminogen activator (rtPA) and mechanical thrombectomy. However, the narrow therapeutic window and extensive exclusion criteria limit the application of these treatments, benefiting only a minority of patients. This underscores the need for continued efforts to improve ischemic stroke therapies4,5. In vitro models are inadequate for replicating the complex pathophysiological responses following a stroke, making animal models an indispensable component of preclinical stroke research. Human focal cerebral ischemia is most frequently caused by thrombotic or embolic occlusion of the middle cerebral artery (MCA), which makes rodent models designed to simulate MCA occlusion (MCAO) highly relevant6.
The filament-induced MCAO model, the most widely adopted in stroke research, facilitates occlusion at the onset of the middle cerebral artery (MCA) and subsequent reperfusion, leading to extensive infarctions across subcortical and cortical areas of the brain. The advantage of this model lies in its ability to restore blood flow after inducing focal ischemia, thereby paralleling the pathophysiological processes observed in human stroke7. Additionally, the model simulates reperfusion injury, a critical factor in the extent of damage8. However, the MCAO model has limitations, including variability in infarct volume, with the standard deviation potentially reaching up to 64% of the mean value in some studies9. Despite over three decades of use, efforts to enhance the model's reliability are ongoing, yet significant variations in ischemic lesion volume persist across studies and laboratories10,11,12.
This article introduces a self-manufactured filament for inducing models evaluating neurological deficit scores and cerebral infarction areas. It examines the correlation between filament lengths coated with silicone and the success and stability of the MCAO model. This production technique yields filaments with commendable consistency, contributing to the development of a relatively stable MCAO model.
Protocol
Representative Results
Discussion
This study demonstrates a simple and cost-effective method for fabricating filament, confirming its feasibility in creating an MCAO model. The length of the filament's silicone coat can be adjusted according to experimental needs, offering additional flexibility. The preparation of a 5 mm filament embolus achieved a 100% success rate without any occurrences of subarachnoid hemorrhage (SAH) in mice. In the group using 10 mm filament emboli, there were instances of SAH, while the rest of the mice showed clear infarctio…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Wu Jieping Medical Foundation (320.6750.161290).
Materials
| 10 mL Syringe | Haidike Medical Products Co., Ltd. | Instrument for making filaments | |
| 2,3,5-Triphenyltetrazolium Chloride (TTC) | Sigma-Aldrich | G3005 | Dye for TTC staining |
| 24-well culture plate | Corning | CLS3527 | Vessel for TTC staining |
| 26 G syringe needle | Haidike Medical Products Co., Ltd. | Instrument for making filaments | |
| 4% paraformaldehyde | Servicebio | G1101 | Tissue fixation |
| 6-0 nylon suture | Haidike Medical Products Co., Ltd. | Materials for making filaments | |
| Anesthesia system for isoflurane | Rwd Life Science Co., Ltd. | R610 | Anesthetized animal |
| Bipolar electrocoagulation generator | Yirun Medical Instrument Co., Ltd. | ZG300 | Equipment for surgery |
| Constant temperature water bath | Spring Instrument Co., Ltd. | HH-M6 | TTC staining |
| Eye ointment | Guangzhou Pharmaceutical | H44023098 | Material for surgery |
| Heat blanket | ZH Biomedical Instrument Co., Ltd. | Maintain body temperatur | |
| Isoflurane | Rwd Life Science Co., Ltd. | R510-22-10 | Anesthetized animal |
| Meloxicam | Boehringer-Ingelheim | J20160020 | Analgesia for animal |
| Microsurgical artery clamp | Shanghai Jinzhong Surgical Instruments Co., Ltd. | W40130 | Instrument for surgery |
| Microsurgical hemostatic clamp forceps | Shanghai Jinzhong Surgical Instruments Co., Ltd. | M-W-0022 | Instrument for surgery |
| Microsurgical instruments set | Rwd Life Science Co., Ltd. | SP0009-R | Equipment for surgery |
| Mouse thermometer | Hubei Dasjiaer Biotechnology | FT3400 | Intraoperative temperature monitoring |
| Pentobarbital sodium | Sigma-Aldrich | P3761 | Euthanized animal |
| Shaver | Joyu Electrical Appliances | PHC-920 | Equipment for surgery |
| Silicone Sealant | Kafuter | K-704 | Materials for making filaments |
| Stereomicroscope | Rwd Life Science Co., Ltd. | 77001S | Equipment for surgery |
| Suture thread with needle (3-0) | Shanghai Pudong Jinhuan Medical Products Co., Ltd. | F404SUS302 | Equipment for surgery |
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