Establishment of a Modified Ferric Chloride-Induced Superior Sagittal Sinus Thrombosis

Summary

This study presents a protocol for establishing a reproducible, minimally invasive rat model of superior sagittal sinus thrombosis induced by ferric chloride, utilizing a silk-suture method in combination with laser speckle contrast imaging and high-resolution ultrasound for evaluation.

Abstract

Cerebral venous thrombosis (CVT) is a rare but potentially life-threatening neurological disorder. Animal models offer critical platforms for investigating their pathophysiology and evaluating potential therapies. In this study, we present a modified rat model of superior sagittal sinus (SSS) thrombosis using a FeCl₃-soaked suture technique. Male Sprague-Dawley rats (220-250 g) underwent a paramedian scalp incision and creation of a 1 cm cranial bone window over the SSS. Compared to the traditional midline incision, this approach prevents adhesion among the incised skin, the bone window, and the underlying dura mater. Additionally, it avoids the interference caused by suture knots located over the SSS, thereby improving the quality of ultrasound imaging. A 2-0 silk suture soaked in 40% FeCl₃ was applied directly onto the exposed sinus for two consecutive 5-min intervals. Compared to the traditional FeCl₃-filter paper method, this technique minimizes direct chemical injury to the parasagittal cortex. Laser speckle contrast imaging was employed to assess cerebral venous perfusion before and after FeCl₃ application. A significant reduction in blood flow confirmed successful thrombosis. Seven days post-surgery, thrombus formation and hemodynamic changes were evaluated via high-resolution ultrasound and analyzed using Vevo LAB software, which provided volumetric and hemodynamic data of the thrombus. This modified model offers a reproducible, minimally invasive, and imaging-compatible approach for preclinical CVT research.

Introduction

Cerebral venous thrombosis (CVT) is a special type of cerebrovascular disease, accounting for 0.5-3% of all strokes and serving as an important cause of stroke in young adults¹. The core pathological change of this disease is thrombosis within the dural venous sinuses and/or cerebral veins, which leads to impaired cerebral venous drainage, subsequently causing increased intracranial pressure, cerebral edema, venous infarction, or hemorrhage². To better elucidate its underlying mechanisms and evaluate novel therapeutic interventions, reliable and reproducible animal models are indispensable. Given that the superior sagittal sinus (SSS) is the most frequently affected site and its superficial location facilitates operational procedures^2,3, current animal experiments primarily utilize the superior sagittal sinus thrombosis (SSST) model.

Among various modeling methods, the ferric chloride (FeCl₃) induction method has become one of the most widely used techniques due to its operational simplicity and cost-effectiveness⁴. In this model, the topical application of a FeCl₃ solution directly damages the venous sinus endothelium, exposing the subendothelial matrix through its strong oxidative properties. This exposure activates platelet aggregation and the coagulation cascade, ultimately inducing venous sinus thrombosis⁵. However, compared with other methods, the traditional FeCl₃-filter paper model also has several drawbacks. The most critical issue is that the paramagnetic properties and high magnetic susceptibility of FeCl₃ generate substantial artifacts during magnetic resonance imaging, severely compromising the accuracy of thrombus assessment and venous sinus recanalization observation⁶. Furthermore, the shape of the filter paper does not fit well with the SSS, making it prone to injuring the brain parenchyma surrounding the sinus. Therefore, it is difficult to determine whether the parenchymal damage around the SSS results from the thrombosis itself or from direct chemical injury caused by FeCl₃.

The overall goal of the present work is to establish a modified FeCl₃-induced SSST rat model that reduces direct cortical injury caused by FeCl₃ and allows for the assessment of blood flow and thrombus size within the SSS. This study introduces a paramedian approach combined with a FeCl₃-soaked suture technique, which minimizes direct cortical chemical injury caused by FeCl₃, and enables real-time, noninvasive, and quantitative monitoring of venous sinus blood flow and thrombus formation using high-resolution ultrasound.

Protocol

This protocol has been approved by the Animal Experiments and Experimental Animal Welfare Committee of Capital Medical University with Approval No. AEEI-2025-138. The reagents and the equipment used are listed in the Table of Materials.

1. Establishment of a modified FeCl₃-induced rat SSST model

1. Use male Sprague-Dawley (SD) rats aged 6-7 weeks, weighing 220-250 g for the experiment. Prior to surgical procedures, acclimate the rats for 1 week and withhold food for 12 h.
2. Prepare the following reagents, instruments, and equipment: 2% pentobarbital solution, 40% FeCl₃ solution, 5% povidone-iodine, 75% alcohol, normal saline, ophthalmic ointment, disposable razor blades, scalpels, surgical scissors, forceps, 2-0 and 4-0 silk sutures, reverse-cutting needles, needle holders, 1-mL and 5-mL syringes, medical tapes, a rodent operating platform, a heating pad with rectal temperature probe, and a micro handheld cranial drill.
3. After weighing, administer preoperative analgesia via subcutaneous injection of buprenorphine (0.1 mg/kg). Following a 15–20 min interval, anesthetize the rat by intraperitoneal injection of 2% pentobarbital (50 mg/kg) (following institutionally approved protocols). Then, pinch the skin of the hind limb with forceps to confirm the absence of withdrawal and pain responses.
4. Apply ophthalmic ointment and shave the rat's scalp hair with a disposable razor blade.
5. Secure the rat in a prone position on a rodent operating platform with medical tapes, and maintain the body temperature at 37 ± 0.2 °C with a heating pad and continuously monitor core temperature with a rectal probe.
6. Disinfect the scalp with alternating rounds of 5% povidone‑iodine, followed by 75% alcohol and repeat three times each.
7. Make a 15-mm paramedian skin incision on the scalp, then bluntly dissect the underlying fascia and periosteum to fully expose the skull.
8. Thin the skull with a cranial drill until the SSS is clearly exposed, starting from the lambda and extending 10 mm anteriorly along the sagittal suture. Employ an intermittent drilling technique to avoid damaging the underlying dura mater and SSS, and repeatedly irrigate the drill bit with normal saline to prevent thermal injury to the cortex.
9. Apply a 10-mm segment of 2-0 silk suture soaked with 40% FeCl₃ solution onto the exposed SSS surface for 5 min, then replace the suture with a newly soaked segment for another 5-min application.
10. Rinse the surgical field with 0.5 mL normal saline three times to remove residual FeCl₃.
11. Close the skin with interrupted sutures using 4-0 silk suture, followed by povidone-iodine disinfection.
12. Observe the rat until fully awake, then return the rat to the cage.

2. Assessment of successful model establishment

NOTE: During the surgical procedure, LSCI was used to measure the venous blood flow in the SSS after creating the cranial window and before FeCl₃ application, and again after FeCl₃ application and rinsing of the surgical field, but prior to skin closure. The model is considered successfully established when the venous blood flow in the SSS shows a significant decrease.

1. Turn on the LSCI and the corresponding software (RFLSI v5.0), and then press online mode button.
2. Adjust the height and position of the instrument until the indicator laser is centered in the field of view.
3. Place the rat on a foam platform and adjust the position until the SSS aligns with the indicator laser.
4. Adjust the magnification and focus until the image displays clearly. Set the lower and upper limits of the pseudocolor threshold to 10 and 200 to optimize the visual appearance of the pseudocolor image.
5. Press the Set ROI button, select the circular tool, and delineate the SSS region.
6. Press the Record button to obtain the venous blood flow and capture both original and pseudocolor images.

3. Assessment of thrombus area/volume and hemodynamic changes

NOTE: Several days to several weeks after the procedure, use high-resolution ultrasound to evaluate thrombus changes and vascular recanalization.

1. Prepare the following drugs, instruments, and equipment: isoflurane, ultrasound gel, disposable razor blades, medical tapes, a small animal anesthesia machine, a high-resolution small animal ultrasound and photoacoustic imaging system, a 70 MHz ultra-high frequency UHF57x linear array transducer, and a 3D-acquisition motor.
2. Install the 3D-acquisition motor and the UHF57x transducer. Then, initialize the motor.
3. Anesthetize the rat in an induction chamber with 3% isoflurane mixed with 1 L/min oxygen.
4. Place the rat in the prone position on the 37 °C thermostatic imaging plate, with the head and limbs gently secured using medical tapes, and maintain anesthesia with 1.5%-2% isoflurane through a nose cone.
5. Shave the scalp hair with a disposable razor blade to expose the detection area, then apply ultrasound gel.
6. Adjust the position and orientation of the rat relative to the transducer until the SSS and intraluminal thrombus are clearly visualized.
7. Acquire sequential tomographic images of the SSS in both sagittal and coronal planes using 3Dimaging in B-mode with a step size of 0.04 mm.
8. Use Color Doppler mode to observe blood flow in the SSS, and then apply Pulsed-Wave (PW) Doppler mode to measure blood flow velocity.
9. Discontinue anesthesia and return the rat to the cage after it is fully awake.
10. Export the images and analyze them using Vevo LAB software. Record the maximum sagittal and coronal cross-sectional areas, and volume of the thrombus in the SSS, along with the maximum blood flow velocity of the sinus.

Results

The schematic diagram of the modified FeCl₃-induced SSST model is shown in Figure 1. Experimental results demonstrated that after application of the FeCl₃-saturated silk suture to the SSS, LSCI detected a significant reduction in local blood flow, indicating successful occlusion of the venous sinus and confirming the sensitivity of LSCI for detecting dynamic perfusion changes (Figure 2). On postoperative day 7, small-animal ultrasound confi...

Discussion

CVT is an uncommon but potentially life-threatening specific type of cerebrovascular disease¹. To further investigate its pathological mechanisms and develop effective intervention strategies, researchers have established various animal models of SSST, primarily including SSS ligation or occlusion^7,8,9, prothrombotic substance injection¹⁰, autologous blood clot injection

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL Syringe	Shenzhen Huayon Biotechnology Co., Ltd.	21-3020
2-0 silk sutures	Shanghai Pudong Jinhuan Medical Products Co.,Ltd	A224
3D-acquisition motor	Fujifilm VisualSonics	https://www.visualsonics.com/gallery/extended-3d-motor
4-0 silk sutures	Shanghai Pudong Jinhuan Medical Products Co.,Ltd	A222
5% povidone-iodine	Shandong Lircon Medical Technology Co., Ltd.	https://www.lircon.cn/products_details/100.html
5 mL Syringe	Shenzhen Huayon Biotechnology Co., Ltd.	21-3023
Disposable razor blade	Dongyang Jinmin Trading Co., Ltd.	ST300
FeCl3	Macklin	I811935
Heating pad	Shenzhen Huayon Biotechnology Co., Ltd.	36-0001
Isoflurane	RWD Life Science	R510-22-10
Laser speckle imaging system	RWD Life Science	RFLSI III
Linear array transducer	Fujifilm VisualSonics	UHF57x
Medical tape	Suzhou Konlida Medical Supplies Co., Ltd.	B02
Micro handheld cranial drill	RWD Life Science	78001
Needle holder	RWD Life Science	F31025-13
Normal saline	Shanghai Beyotime Biotechnology Co., Ltd.	ST341-500ml
Ophthalmic ointment	Shenzhen Huayon Biotechnology Co., Ltd.	19-7223
Reverse-cutting needles	Shanghai Pudong Jinhuan Medical Products Co.,Ltd	TJ-3-514
Rodent operating platform	Shanghai Yuyan Scientific Instrument Co., Ltd.	30150
Scalpel blade	RWD Life Science	S31015-01
Scalpel handle	RWD Life Science	S32003-12
Small animal anesthesia machine	RWD Life Science	R500
Small animal ultrasound and photoacoustic imaging system	Fujifilm VisualSonics	Vevo F2
Smooth Forceps	RWD Life Science	F12013-10
Sodium Pentobarbital	Sigma-Aldrich (Shanghai) Trading Co., Ltd.	P3761
Surgical Scissors	RWD Life Science	S14014-10
Toothed Forceps	RWD Life Science	F12011-13
Ultrasound gel	Shandong Lircon Medical Technology Co., Ltd.	https://www.lircon.cn/products_details/230.html