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

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.

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, 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, 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 5% povidone-iodine.
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.

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 confirmed the formation of a thrombus in the SSS with partial luminal recanalization (Figure 3). The Vevo LAB software was used to analyze ultrasound images, obtaining cross-sectional areas (in sagittal and coronal planes) and the volume of the thrombus, as well as blood flow velocity in the residual lumen (Figure 3 and Figure 4).

These results collectively demonstrate the capability of the combined FeCl3-soaked suture and imaging approach to reliably establish, monitor, and quantify SSS thrombosis in vivo. LSCI enables the real-time assessment of venous perfusion immediately following FeCl3 exposure, whereas high-resolution ultrasound allows for the longitudinal evaluation of thrombus morphology and hemodynamic alterations without the need for invasive sampling. This multimodal strategy provides a comprehensive characterization of both the acute occlusive process and the subsequent hemodynamic recovery.

Figure 1: Schematic diagram of the modified FeCl3-induced SSST model. (A) Paramedian incision (black arrow). (B) Skull drilling area (red line). (C) 2-0 silk suture saturated with FeCl3 solution (black line). Please click here to view a larger version of this figure.

Figure 2: Blood flow in the SSS before and after local application of FeCl3. Black arrows indicate the SSS. In the pseudocolor pattern, red/yellow represents high perfusion areas, while blue/green indicates low perfusion regions. Please click here to view a larger version of this figure.

Figure 3: Thrombus formation in the SSS with partial recanalization. (A) Maximum sagittal cross-sectional area (B-mode). (B) Maximum coronal cross-sectional area (B-mode). (C) Maximum blood flow velocity (PW Doppler mode). Red, blue, and yellow arrows indicate thrombus, residual lumen, and blood flow, respectively. Please click here to view a larger version of this figure.

Figure 4: Three-dimensional volume measurement of the SSST. Image acquisition slice thickness: 0.04 mm. Please click here to view a larger version of this figure.

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 injection¹¹, photochemical induction¹², and FeCl₃ induction⁴. Among these, the SSS ligation or occlusion model cannot simulate the spontaneous dissolution process of thrombi, making it unsuitable for therapeutic efficacy evaluation¹³; Prothrombotic substance injection carries the risk of drug diffusion into the systemic circulation, potentially causing non-target site thrombosis⁸; Although autologous blood clot injection achieves embolism, it does not involve core pathological mechanisms of venous thrombosis such as vascular endothelial injury, hemodynamic abnormalities, and hypercoagulability¹¹; Photochemical induction is more suitable for small vessel thrombosis modeling, as the high blood flow velocity in the SSS affects its thrombus induction efficacy⁴.

In 2005, Röttger et al. first established the FeCl₃-induced SSST model in rats⁴. This model successfully induced thrombus formation by applying filter paper strips saturated with 40% FeCl₃ solution to the surface of the SSS for 4 min, and observed the spontaneous dissolution of thrombi⁴. Subsequent studies have mostly adjusted the concentration of FeCl₃ (ranging from 10% to 40%) or the exposure duration (from 5-15 min) based on this foundation^6,14,15. We made the following improvements to this model: First, the traditional midline incision was modified to a paramedian incision, which effectively prevents postoperative adhesion among the skin, bone window, and dura mater, while eliminating interference from suture knots above the SSS on imaging signals, thereby significantly improving the quality of ultrasound imaging. Second, a smaller-diameter 2-0 silk suture was used to replace the filter paper strips, minimizing the extent of chemical damage to the cortical tissue adjacent to the sinus. In addition, LSCI was employed to compare changes in local blood flow in the SSS before and after FeCl₃ application, in order to assess whether the model was successfully established. With advantages including non-invasiveness, operational simplicity, and real-time imaging, LSCI has been widely applied in dynamic CBF monitoring for various stroke models, including SSST¹⁶. Most importantly, we innovatively used the small-animal ultrasound imaging system to perform 3D scanning of the SSS, enabling direct observation of the thrombi. Current researches predominantly rely on magnetic resonance venography or digital subtraction angiography to evaluate luminal recanalization^3,17. However, these techniques can only indirectly infer the presence of thrombi or vascular recanalization through changes in blood flow signals. In contrast, ultrasound technology enables the direct visualization of thrombi and allows for the precise quantification of their cross-sectional area, volume, and intraluminal blood flow velocity, thereby providing a more comprehensive and objective basis for evaluating the efficacy of different treatment approaches in thrombus dissolution.

In summary, by integrating with LSCI and high-resolution ultrasound imaging technologies, the modified FeCl₃-induced SSST model described in this study provides a more reliable and intuitive experimental platform for the development and efficacy evaluation of treatment measures. However, several limitations should be acknowledged. First, the model does not permit direct observation of thrombosis or changes in blood flow in the bridging veins adjacent to the superior sagittal sinus. Second, FeCl₃ induces endothelial injury through strong oxidative stress, which might not fully mimic the complex pathophysiological processes of spontaneous CVT in humans. Third, this model primarily reflects the acute and subacute phase of thrombosis, while the chronic remodeling processes are not well represented.

Future studies may extend this method to explore venous remodeling and neurovascular recovery following CVT, or to evaluate the efficacy of novel thrombolytic and neuroprotective agents under longitudinal imaging guidance. The integration of additional modalities, such as photoacoustic imaging or molecular contrast-enhanced ultrasound, could further enable visualization of thrombus composition, inflammation, and neovascularization. Moreover, adapting this minimally invasive approach to larger animal models may facilitate translational research and preclinical testing of endovascular or pharmacological interventions for cerebral venous thrombosis.