A Simplified Operation for the Endovascular Perforation Murine Model of Subarachnoid Hemorrhage

Summary

Here we present a simplified operation for the endovascular perforation murine model of subarachnoid hemorrhage combined with neurological scoring.

Abstract

The endovascular perforation model is commonly utilized as a method to simulate subarachnoid hemorrhage (SAH) in experimental studies. Comprehensive and reliable as it is, it requires complicated techniques. This protocol simplifies the model in some detail, combined with neurological scoring. We use a nylon suture to loop and pull the common carotid artery (CCA) to block blood flow temporarily other than traditional ligation, which offers a clear operative field. The utilization of the electrocautery pen to fuse the blood vessels reduces the risk of bleeding. Moreover, we use a filament with a black mark to make it easy to determine the depth of the puncture.

The unimpeded advancement of the black marker past the carotid bifurcation, where the common carotid artery (CCA) diverges into the internal carotid artery (ICA), accompanied by minimal resistance, indicates that the filament has successfully traversed to the intracranial junction of the anterior cerebral artery (ACA) and middle cerebral artery (MCA). This phenomenon signifies the filament's positioning at a critical cerebrovascular convergence point. Then, slightly advancing the filament forward to puncture the blood vessel allows easier operation for endovascular perforation in mice, thereby facilitating the application of the endovascular perforation model in genetically modified mice. This can be very crucial for molecular research and pharmaceutical research and development.

Introduction

Subarachnoid hemorrhage (SAH), a severe subtype of stroke, is linked to high rates of morbidity and mortality, especially affecting people in their mid-fifties. Spontaneous SAH is mainly caused by the rupture of intracranial aneurysms, which predominantly occurs in the circle of Willis^1,2. Clinically, patients often present with thunderclap headaches, neurological dysfunction, and a rapid decline in consciousness³. Despite advances in neurocritical care, nearly 40% of patients die within a month after bleeding⁴. Several secondary complications can occur after ane....

Protocol

C57BL/6 male mice (6-8 weeks old), free of specific pathogens, were kept in an animal facility with regulated temperature and acclimatization conditions, maintained on a 12-hour light/dark cycle. The mice were provided with unlimited access to a standard rodent diet and sterilized tap water. A 2 week acclimatization period was allowed before the start of the experiments. All procedures involving animals were approved by the Animal Ethics Committee of Longhua Hospital and conducted in accordance with the National Science and Technology Committee of China's guidelines for the care and use of laboratory animals.

1. Animal preparation....

Results

Neurological scores
Nine mice of each group were assessed 24 h postoperatively using a modification of the scoring system reported by Sugawara et al.¹¹ to evaluate SAH. The maximal obtainable score is 18. Among them, n = 9 animals in the sham group all scored 18. In the SAH group, the average score was 11 (p < 0.0001) (Figure 2).

Pathological changes in the brain
Blood clots were visible on the b.......

Discussion

To conclude, this study presents a simplified and reliable murine model of subarachnoid hemorrhage (SAH) induced by endovascular filament insertion, offering a convenient and effective approach. Compared with the existing endovascular perforation model, this protocol is the same in the perforation step: The filament is introduced into the external carotid artery (ECA), advanced through the common carotid artery (CCA), and subsequently directed into the internal carotid artery (ICA), where it is advanced further to perfor.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6 mice	Shanghai Jiesjie Laboratory Animal Co., LTD
electrocautery pen	Zhuoyu Angel Medical Equipment Co., Ltd	GermanV50	Suitable for use in superficial minor surgeries, tissue coagulation, and cauterization of vegetation.
isoflurance	RWD	R650-IE	Isoflurane is an inhaled general anesthetic. It has a small blood/gas partition coefficient. When used for animal anesthesia, it can induce anesthesia smoothly, rapidly and comfortably, with a quick recovery, good muscle relaxation, and no excitatory effect on the sympathetic nervous system. The metabolic rate of isoflurane in the liver is low, so it has little toxicity to the liver, and there are no obvious side effects even with repeated use. To avoid the adverse effects of anesthetic waste gas on the environment and laboratory personnel, it is recommended to use it together with a gas recovery system.
MACO filament	Beijing Cinontech Co.LTD	1620-A2	Mostly used for mice weighing 20-25 g, the suture tip is melted into a hemispherical shape, without polylysine or silicone coating, marked 9-10 mm away from the tip, disinfected, and can be used directly. This model has three packaging specifications. It is recommended to conduct a pre-experiment before the formal experiment to determine the appropriate model.
microscope	Olympus	BX61VS	A fully motorized upright microscope equipped with the UIS2 optical system, designed for high-resolution and high-precision imaging. Features include motorized nosepiece, stage, and focus, as well as virtual slide capabilities for creating large, high-resolution digital images of specimens. Suitable for advanced research applications in pathology, cell biology, neuroscience, and material science.
nylon suture	Huawei Medical Supplies Co., Ltd	J1110	A non-absorbable surgical suture made from high-strength, biocompatible materials.