A Modified Mouse Model of Intracranial Aneurysm Based on Hemodynamic Change and Hypertension

Summary

Intracranial aneurysm (IA) was constructed in mice using the risk factors of hypertension and hemodynamic changes. Hemodynamic changes were induced by ligating branches of the carotid artery, while hypertension was achieved by ligating the posterior branches of the renal artery. IA formation was detected through magnetic resonance angiography, stereomicroscopy, and pathological analysis.

Abstract

Intracranial aneurysm (IA) poses a significant health risk due to morbidity and mortality associated with aneurysm rupture. However, the molecular mechanisms underlying IA development remain unclear, and a suitable mouse model is required. A mouse model of IA was established by ligating the pterygopalatine artery (PPA) to induce additive hemodynamic changes, combined with hypertension induction. In C57BL/6 male mice, vessels, including the right PPA, external carotid artery (ECA), occipital artery (OcA), and the left contralateral common carotid artery (CCA), were ligated to induce hemodynamic changes. One week later, the bilateral posterior branches of the renal artery (pRA) were ligated, and an 8% salt diet was introduced to induce hypertension. Magnetic resonance angiography (MRA), stereomicroscopy, and immunohistochemical (IHC) staining were performed to evaluate the morphological and pathological changes in IA three months after induction. In the experimental group, four mice died after the initial induction. IA at different locations was detected in five of the eleven remaining mice. Both microscopic and MRA examinations confirmed IA formation. Pathological and IHC analyses revealed disruption of the internal elastic lamina, disconnection of collagen fibers, and infiltration of CD86-positive M1 macrophages, findings consistent with those observed in human IA. This mouse model of IA replicates the pathological changes observed in human samples and may serve as a valuable tool for investigating the molecular mechanisms of IA formation and progression.

Introduction

The prevalence of intracranial aneurysm (IA) is estimated to be 3.2% of the general population¹. IA poses a significant health risk due to its high associated morbidity and mortality. IA is a complex and multidimensional pathological condition influenced by hemodynamic changes, inflammation, and vascular remodeling^2,3. Hemodynamic changes and hypertension are implicated in the formation and progression of aneurysms^4,5. IA frequently occurs at cerebral bifurcations with elevated hemodynamic shear stress⁶, and bifurcations with narrow angles are identified as risk factors for IA development in humans⁷. Despite advancements in endovascular treatments and surgical strategies, subarachnoid hemorrhage caused by IA rupture remains catastrophic. Therefore, exploring pharmacological treatments is a promising approach for preventing aneurysm rupture⁸. However, the mechanisms underlying the pathological formation and progression of IA remain unclear. Developing a suitable mouse model for IA formation and progression, based on human risk factors, is crucial to uncovering the underlying mechanisms and identifying potential therapeutic targets. This study aims to construct a model of IA formation without rupture in mice that mimic human IA characteristics.

The circle of Willis (CW) connects and communicates the right internal carotid artery (ICA), left ICA, and bilateral vertebrobasilar arteries. The CW serves as a compensatory mechanism in cases of occlusion or stenosis of the ICA or vertebral artery⁹. The pterygopalatine artery (PPA) is a branch of the ICA that supplies blood to the external part of the brain¹⁰. Based on the compensatory function of the CW, PPA occlusion increases blood flow in the ICA. Combining ligation of the left common carotid artery (CCA), right external carotid artery (ECA), and occipital artery (OcA) results in increased blood flow in the CW, particularly at narrowed angles, leading to hemodynamic changes. In this model, the blood supply to the brain is supported by the vertebrobasilar artery and the right ICA. PPA ligation did not contribute to mortality in the mice¹¹.

To induce an IA model based on elastase injection, hypertension was induced by angiotensin-II (Ang-II) release via an Alzet pump or deoxycorticosterone acetate (DOCA)-salt^12,13. The high cost of Alzet and DOCA should be considered in experiments involving a large number of animals. The achieved levels of hypertension were not significantly different between ligation of the posterior and inferior branches of the bilateral renal arteries or only the posterior branches of the bilateral renal arteries. However, the former approach resulted in greater renal dysfunction¹⁴. Therefore, ligation of the bilateral posterior renal arteries (pRA) is considered a rational method for most investigators.

Elastase was injected into the cerebrospinal fluid at the right basal cistern via a single stereotaxic injection¹². The elastase injection-based IA model caused 60%-80% IA rupture three weeks after injection^15,16, which is too short to study IA formation and development. Furthermore, there is no evidence to suggest elevated elastase levels in humans during IA formation. Additionally, stereotaxic injection into the right cistern is associated with high mortality and disability in mice, posing significant challenges for novices.

In this study, a mouse model of IA without rupture within three months was constructed based on human risk factors. This model eliminates the high cost associated with DOCA and Alzet. Moreover, it can be performed using only a stereomicroscope and can be easily mastered by novices.

Protocol

All operational procedures in mice adhered to the criteria of the Ethical Review Committee and were approved by the Institutional Animal Care and Use Committee of Shanghai Jiaotong University. C57BL/6 male mice (8 weeks old, 20-25 g) were housed at a temperature of 22 °C with a 12 h/12 h light/dark cycle. The operational process is shown in Figure 1A. Briefly, in anesthetized animals, the left common carotid artery (CCA), right external carotid artery (ECA), occipital artery (OcA), and pterygopalatine artery (PPA) were ligated to induce hemodynamic changes. Hypertension was subsequently induced by ligating the bilateral renal arteries (bRA) one week after the initiation of hemodynamic changes, and the animals were fed a diet containing 8% salt. Hemodynamic changes and IA formation are depicted in Figure 1B. Details of the reagents and equipment used in this study are provided in the Table of Materials.

1. Establishment of mouse UIA based on hemodynamic changes and hypertension

NOTE: Mice were fasted for 12 h before the operation. Surgical instruments were sterilized by soaking them in 70% alcohol for at least 30 min.

1. Administer anesthesia (following institutionally approved protocols) using a small animal anesthesia machine with 2% isoflurane inhalation in a mixture of O₂ (1 L/min) on a heated pad maintained at 37 °C.
2. Ligate the left CCA
   1. Make a 1 cm linear incision along the cervical midline. Dissect subcutaneous tissue and platysma to expose the trachea.
   2. Pull the jugular vein away and locate the carotid sheath along the trachea (Figure 2A i,ii). Separate the left CCA from the vagus nerve. Ligate the left CCA with a 6-0 silk suture (Figure 2A iii,iv).
3. Ligate the right ECA and OcA
   1. Expose the anatomical structure of the CCA, ICA, ECA, OcA, vagus nerve, and hypoglossal nerve on the right side. Ligate the right ECA with a 6-0 silk suture (Figure 2A iii,iv).
   2. Isolate the OcA and ligate it with an 8-0 silk suture (Figure 2A v - viii). Disconnect the OcA using two straight micro forceps.
      NOTE: The OcA, arising proximally from the ECA, lies below the hypoglossal nerve (Figure 2A v - viii). Dissecting the OcA helps to expose the PPA anatomy effectively.
4. Ligate the right PPA
   1. Isolate the hypoglossal nerve from the ICA. Place surgical cotton between the hypoglossal nerve and ICA to protect the nerve from procedure-related injury.
   2. Dissect the perivascular tissue around the PPA using microforceps. Clamp the PPA and temporarily pull it up lightly. Place an 8-0 silk suture between the PPA and ICA to ligate the PPA (Figure 2A ix,x).
   3. Ligate the PPA and maintain sufficient distance between the ligation site and the origin of the PPA to ensure blood flow in the circle of Willis (Figure 2A x).
      NOTE: The primary challenge in this step is ligating the PPA within an extremely tight operating space without impairing peripheral nerves and vascular structures. Avoid compressing the trachea during PPA exposure.
5. Finalize the operation
   1. Sterilize the operational field with povidone-iodine and suture the wound. Monitor mice until they recover from anesthesia.
6. Induce hypertension
   1. Make a 2 cm long midline incision at the 12^th vertebral level on the back. Cut the dorsal muscles to expose the kidney. Clamp the adipose tissue around the kidney using forceps and fix the kidney inside the muscle incision (Figure 2B i,ii).
   2. Isolate the adipose tissue around the renal pedicle. Identify the pRA in close contact with the renal vein (Figure 2B iii,iv). Clamp the pRA and pull it up using straight micro forceps. Dissect the fascia between the pRA and renal vein using another micro forceps (Figure 2B v,vi).
      NOTE: Exercise caution to prevent damage to the renal vein, which could lead to catastrophic bleeding.
   3. Ligate the pRA with a 6-0 silk suture (Figure 2B vii,viii). Immediately after ligation, observe ischemic foci forming in the upper part of the kidney (Figure 2B ix,x). Suture the muscle and skin incisions.

2. IA Examination via MRA and stereomicroscope

1. Perform time-of-flight (TOF) 7.0 T magnetic resonance angiography (MRA) three months after aneurysm induction to evaluate IA formation.
   1. Euthanize the mice via exsanguination and cervical dislocation after isoflurane anesthesia (following institutionally approved protocols). Detect IA under a stereomicroscope as previously reported¹⁷.
   2. Infuse the samples with cooled PBS, followed by 4% paraformaldehyde, and then Microfil to visualize vessels and aneurysms.
   3. Define an aneurysm as outward bulging 1.5 times larger than the parent artery, as observed by two independent neurosurgeons^8,18.

3. Histological and immunohistochemistry analyses

1. Isolate the circles of Willis under a microscope. Fix the specimens with 4% paraformaldehyde at 4 °C for 24 h¹⁹.
2. Process the specimens into paraffin and frozen sections for histological and immunofluorescence staining. Stain paraffin sections with EVG and Masson stains according to the manufacturers' instructions¹⁹.
3. Incubate the paraffin sections with a blocking solution to minimize nonspecific binding.
4. Incubate sections with primary antibodies against CD86 overnight at 4 °C. After incubation with a secondary antibody, develop a brown color by reacting the sections with DAB²⁰.
5. Perform counterstaining with hematoxylin and mount the sections with an aqueous mounting solution²⁰.

Results

Rate of IA formation
In the experimental group (n = 15), 2 mice died within the first week after the initial procedure for unknown reasons. One mouse died from an infection in the back wound on the third day after the second procedure, and another mouse died on day 38 for unknown reasons, with no aneurysms detected. In the control group (n = 5), all 5 mice survived until sacrifice. Among the surviving mice in the experimental group (n = 11), the systolic blood pressure at three months post-induction ...

Discussion

This study presents a modified approach to constructing a mouse model of IA through ligation of the PPA to induce additive hemodynamic changes in combination with hypertension. MRA imaging and microscopic analysis demonstrated significant aneurysmal changes in the circle of Willis. The pathological alterations observed in this model are consistent with those found in human samples. This mouse model may serve as a valuable tool for investigating the molecular mechanisms underlying IA formation and developme...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
7.0 T magnetic resonance angiography	Bruker	BioSpec 70/20
C57BL/6 mice	Charles River Laboratories		sex: male
CD86 antibody	CST	91882
Elastic Van Gieson (EVG)stain	Solarbio	G1597
Masson	Solarbio	G1340
Micro forcep	Shanghai Jinzhong Instrument Company
Microfil	Flow Tech Inc.	MV-120
Small animal anesthesia machine	RWD	R500
Stereo microscope	Shanghai Optical Instrument Company	XYH-6B
Suture	Shanghai Jinhuan Medical Company		6-0, 8-0