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 aneurysmal SAH, which leads to progressive neurological deterioration. However, the pathophysiology and clinical characteristics of SAH are still poorly understood.

Thus, to better understand the underlying mechanisms and improve the therapeutic strategies, comprehensive and reliable models in small animals are indispensable. To date, numerous protocols for establishing SAH models have been put forward. For instance, one approach involves injecting autologous blood into the cisterna magna. Another modified protocol entails two separate injections, with one into the cisterna magna and the other into the optic chiasm cistern. The autologous blood injection has gained widespread adoption due to its technical simplicity and favorable mortality rate^5,6. To achieve the possibility of using a smaller and less expensive laboratory animal, Barry et al. described the first SAH model on rats in 1979 that involved puncturing the basilar artery with tungsten microelectrodes after removing the skull⁷.

In 1995, another endovascular perforation model, a modified version of the Zea-Longa model of cerebral ischemia, was presented, whose present form later proved to be the most commonly used model to study SAH⁸. Previous research has demonstrated that mice and humans possess a similar intracranial vascular structure, referred to as the circle of Willis, where the intracranial aneurysm predominantly occurs⁹. This non-craniotomy method replicates human aneurysm rupture mechanisms through precise ICA bifurcation perforation¹⁰. Furthermore, the genetically modified mouse is frequently used in the study of molecular mechanisms. However, due to its complicated techniques in small animals, the endovascular perforation model is predominantly utilized in rats or rabbits whereas its application in mice is comparatively less common. Therefore, it is of great significance to simplify the endovascular perforation murine model and reduce its high mortality rate.

Here, we present a simplified modification of the endovascular perforation murine model, integrating a modified version of the Garcia neurological assessment scale¹¹. Our modification provides an easy, convenient, and reliable model for future experimental studies of SAH.

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

1. Place the mouse on a heating pad preheated to 37 °C and continue to maintain this temperature until the surgery is completed.
2. Anesthetize the mouse by inhalation of 2-2.5% isoflurane. Maintain with 1-1.5% isoflurane during the operation at a flow rate of 0.4-0.6 L/min.
3. During the surgery, apply eye ointment to both eyes to ameliorate dryness of the eyes.
4. Position the mouse on the surgical table, stabilize its head, keep it in a supine position, and secure its limbs with tape.

2. SAH induction

1. Incise the skin with a scalpel blade along the middle of the anterior neck (Figure 1A).
2. Dissect the connective tissue and expose the left common carotid artery (CCA) and its bifurcations.
   NOTE: Protect the vagus nerve and the adjacent glands cautiously (Figure 1B).
3. Loop the CCA with 6-0 nylon suture and leave the two ends free without ligation (Figure 1C).
4. Connect both ends of the suture to the tape and gently pull the suture downward and to the right at a 45° angle to the horizontal plane. Secure the tape to the operating table to temporarily block the CCA (Figure 1D).
   NOTE: When the blood flow within the occluded vessel becomes thread-like, it indicates successful occlusion.
5. Ligate the external carotid artery (ECA) with a nylon suture (Figure 1E).
   NOTE: The cauterization site should be as close to the upper end as possible, with a length of approximately 2 mm from the carotid bifurcation.
6. Fuse the ECA distal to the site of ligation using an electrocautery pen (Figure 1F).
7. Pull the ECA downwards slowly until the internal carotid artery (ICA) is exposed. Align the ECA and the ICA in a straight line, ensuring the proper position for the next procedure (Figure 1G).
8. Sharpen the top of the filament. A mark point on the filament is located 8 mm from the top.
9. Make a small incision with scissors for the filament insertion into the ECA. Insert the filament using a pair of forceps (Figure 1H).
10. Advance the filament carefully with forceps until the black mark completely passes through the bifurcation of CCA and ICA. Slightly advance 2 mm to puncture the blood vessel (Figure 1I).
    NOTE: Significant resistance is felt, which indicates that the anterior cerebral artery (ACA)-middle cerebral artery (MCA) bifurcation has been reached.
11. Retract the filament immediately following perforation.
12. Fuse the ECA using an electrocautery pen (Figure 1J).
13. Remove the tape and withdraw the nylon suture of the CCA to restore the blood flow. Observe the obvious pulsation of the CCA (Figure 1K).
14. Close up the neck incision with 5-0 absorbable sutures.

3. Sham group

1. In the sham group, perform the same surgical operation except that the filament is advanced partially in the ICA and withdrawn without puncturing¹².

4. End of experiment

1. Twenty-four hours post-surgery, evaluate the neurological performance according to the modified version of the scoring system reported by Sugawara et al.¹¹(from the original Garcia et al.¹³). Perform the following six assessments in a blinded manner: spontaneous activity, movement of all limbs, forelimb movement, climbing the wire cage wall, response to touch on both sides of the trunk, and reaction to vibrissae stimulation. Evaluate neurological function using the behavior scoring table (Table 1).
2. Anesthetize the mouse with inhaled isoflurane. Place the mouse in a supine position and secure it to the surgical table with tape, fixing its limbs. Then, use scissors to make an incision along the midline of the abdomen, cut through the abdominal wall, and expose the thoracic cavity. Carefully cut the sternum to expose the heart.
3. Carefully insert an infusion needle into the left ventricle, ensuring the needle is stable and does not penetrate the heart.
4. Begin perfusing with precooled 1x PBS (4 °C) at an appropriate flow rate (approximately 10 mL/min) until the outflowing liquid becomes clear and transparent, indicating that the blood has been fully replaced.
5. Once perfusion is complete, remove the infusion needle and stop the fluid infusion. Proceed with brain tissue collection.

5. Postoperative management

1. When closing up the skin, administer a pain-relieving drug (such as meloxicam, 1-2 mg/kg, subcutaneous injection, every 24 h) to the mice to alleviate the pain.
2. After the surgery, based on the amount of blood loss during the surgical process, inject 0.2-0.4 mL of saline into the mouse intraperitoneally for fluid replacement.
3. Check the animals regularly for spontaneous breathing during the first hours after surgery and monitor twice daily for predefined humane endpoints. Euthanize the mice with CO₂ immediately if they are found to have breathing problems caused by the surgery or meet any endpoints, including >20% weight loss from baseline, sustained inability to access food/water, or severe neurological deficits.
   NOTE: The animal is not left unattended until it has regained sufficient consciousness to maintain sternal recumbency and is not returned to the company of other animals until fully recovered.

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 base of the brain and had accumulated at the circle of Willis, whereas no such findings were detected in the sham group (Figure 3).

Figure 1: Step-by-step images of surgical technique. (A) Open the skin along the middle of the anterior neck. (B) Exposure of the left common carotid artery reveals the 'Y'-shaped junction formed by the common carotid artery, external carotid artery, and internal carotid artery. (C) Pass a suture under the CCA. (D) The CCA is interrupted by a suture. (E) Ligate the ECA with a nylon suture. (F) Fuse the ECA distal to the site of ligation. (G) Pull the ECA downwards. Align the ECA and the ICA in a straight line. (H) Make a small incision into the ECA and insert the filament. (I) Advance the filament carefully until the black mark completely passes through the bifurcation of CCA and ICA and slightly advance forward to puncture the blood vessel. (J) Retract the filament and fuse the ECA. (K) Reopened the CCA to allow reperfusion. Abbreviations: ACA = anterior cerebral artery; CCA = common carotid artery; ECA = external carotid artery; MCA = middle cerebral artery; ICA = internal carotid artery; PPA = pterygopalatine artery. Please click here to view a larger version of this figure.

Figure 2: Neurological scores. The graph shows neurological scores of mice 24 h after SAH. The scores are profoundly decreased in the SAH group compared with the sham group (p < 0.0001). Sham n = 9, SAH n = 9. Abbreviation: SAH = subarachnoid hemorrhage. Please click here to view a larger version of this figure.

Figure 3: Mouse brain anatomy and macroscopic images of Sham and SAH. (A) Schematic illustration of the mouse vascular anatomy, highlighting the site of the filament perforation. (B) Images of the brain base in the simplified subarachnoid hemorrhage surgical group and the sham surgery group. Prior to brain removal, the mice underwent cardiac perfusion with 1x PBS. Compared to the sham group, the surgical group displayed distinct blood clots at the brain base, with notable accumulation around the circle of Willis. Figure 3A was modified from Liu et al.¹⁴.
Abbreviations: SAH = subarachnoid hemorrhage; ACA = anterior cerebral artery; CCA = common carotid artery; ECA = external carotid artery; MCA = middle cerebral artery; ICA = internal carotid artery; PPA = pterygopalatine artery. Please click here to view a larger version of this figure.

Test	0 points	1 point	2 points	3 points
Spontaneous activity (in cage for 5 min)	No movement	Barely moves	Moves but does not appproach 3 walls of cage	Moves and approahes ≥ 3 walls of cage
Spontaneous movement of all limbs	No movement	Slight limb movements	Moves all limbs slowly	Moves all limbs same as pre-SAH
Movement of forelimbs (outstretching when held by tail)	No outreaching	Slight outreaching	Outreach is limited and less than pre-SAH	Outreach same as pre-SAH
Climbing wall of wire cage	Falls from slope	Fails to climb	Climbs weakly	Normal climbing
Reaction to touch on both sides of trunk		No response	Weak response	Normal response
Response to vibrissae touch		No response	Weak response	Normal response

Table 1: Modified Garcia score. Abbreviation: SAH = subarachnoid hemorrhage. This table was reported by Sugawara et al.¹¹.

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 perforate the anterior cerebral artery (ACA). We use a suture to pull the CCA to block blood flow temporarily other than ligation. Unlike rats, the operative field in mice is quite limited. Using traction instead of ligation or using an artery clamp can adequately expose the surgical field. On the one hand, this approach can effectively minimize the incision area required in the neck, thereby reducing trauma to the mouse. On the other hand, it provides a clear operative field and saves substantial time used in ligation, which leads to a shorter procedure time. If the blood flow is occluded for a long period, it may lead to complications such as cerebral ischemia, as well as insufficient bleeding at the puncture site. Another possible problem is uncontrollable bleeding. In this case, we used the electrocautery pen to directly fuse the ECA after pulling out the filament.

In this approach, we used fine filaments made of Polyamide (PA) for perforation instead of Polytetrafluoroethylene (PTFE) tubing. During the procedure, difficulties can arise when the perforating material is either too soft, leading to challenges in perforation, or too rigid, which could potentially cause damage to the vessel. The filament we use has been extensively validated and is designed with an optimal balance of flexibility and rigidity. Additionally, the company marks the filament, with the distance from the tip to the marking point approximately equal to the distance between the bifurcation of the external carotid artery (ECA) and internal carotid artery (ICA) to the bifurcation of the anterior cerebral artery (ACA) and middle cerebral artery (MCA). Furthermore, based on the physiological structure of mice, the filament material is designed with a certain curvature to facilitate perforation, a feature lacking in standard PTFE tubing. Additionally, PA offers a more cost-effective alternative compared to PTFE^15,16.

Inappropriate depth of filament insertion can lead to either insufficient perforation or stroke and/or ICH. Therefore, we use a filament with a black mark located 8 mm from the top to make it easy to determine the depth of the puncture. When the black mark completely passes through the bifurcation of CCA and internal carotid artery (ICA), it indicates that the filament has reached the anterior cerebral artery (ACA)-middle cerebral artery (MCA) bifurcation. Resistance is felt after reaching the ACA-MCA bifurcation, and the force used for perforation should also be appropriate. If the force is excessive, the whole arterial tree moves, which may lead to subdural hematoma instead of SAH¹⁷. Based on our experience, the first advance of the filament is equally important. If it fails, a significant increase in the puncture resistance can be felt, indicating vessel constriction. This makes the subsequent advance of the filament even harder. In this sense, the success rate of this SAH model is related to the experience of the surgeon.

To date, different methods have been proposed for the postoperative evaluation of SAH, including neurological scoring, intracranial pressure (ICP) monitoring, and various radiological imaging techniques, such as MRI grading^18,19. MRI scanning in animal models is time-consuming and not always available to laboratories. In this protocol, the modified Garcia Neurological scoring was used to assess the SAH neurological deficits¹¹. To date, as a composite score system incorporating different dimensions of neurological functions, the modified Garcia Neurological scoring has been commonly employed for assessing functional deficits in rodent models after SAH. This method is non-invasive and provides accurate diagnosis through multiple aspects of neurological function scoring, including spontaneous movement, forelimb movement when the tail is held, and cage climbing^11,20,21. This SAH grading system is straightforward, practical, and simple to apply in experimental models. Among the different methods for inducing SAH, the endovascular perforation model and the injection of autologous blood into the cisterna magna are commonly utilized. The autologous blood injection is easy to perform and carries a low mortality rate, making it the preferred choice^22,23. However, the endovascular perforation model has a mortality rate of about 50%, which is relatively high, failing to reach the standard for an ideal animal model. Despite its deficiencies, the endovascular perforation model avoids the need for a craniotomy while accurately replicating the mechanisms of aneurysmal rupture in humans and producing more profound pathophysiological and histological changes to the brain and cerebral vessels^24,25. Moreover, compared to blood injection models, this technique achieves closer imitations to the changes in blood-brain barrier permeability and higher incidences of vasospasm through filament perforation^26,27. Thus, this model is the closest imitation of human SAH theoretically.

In summary, this protocol simplifies the techniques to establish a murine model of SAH and thereby enhances the wider utilization of the endovascular perforation model in the genetically modified mouse. This is critically important for molecular research and pharmaceutical research and development.

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.