Novel Rat Model of Severe Cerebral Venous Sinus Thrombosis Established by Combination of Semi-Ligation, Ferric Chloride, and Thrombin

Summary

The novel rat model of severe cerebral venous sinus thrombosis (CVST), which is constructed by semi-ligation combined with ferric chloride and thrombin, can more effectively mimic the pathophysiological mechanism of severe CVST in humans.

Abstract

Cerebral venous sinus thrombosis (CVST) is a distinct type of stroke that predominantly affects young individuals, particularly pregnant women. Approximately 60% of CVST patients develop venous cerebral infarction or hemorrhage, which is defined as severe CVST. Currently, various methods are employed both domestically and internationally to induce CVST in animal models. However, these models fail to fully replicate the pathophysiological mechanisms underlying severe CVST, thereby limiting basic research on this condition. A novel rat model of severe CVST can be established through semi-ligation in combination with ferric chloride and thrombin. Semi-ligation was achieved by measuring the cerebral blood flow (CBF) in the region of interest (ROI) before and after ligation of the superior sagittal sinus (SSS) using a Perfusion Speckle Imager (PSI). Semi-ligation of the SSS induces blood stasis within the venous sinus. The topical application of ferric chloride on the surface of the SSS leads to endothelial injury, while direct injection of thrombin into the sinus creates a localized hypercoagulable state. This approach effectively mimics the three key components of thrombosis formation: stasis, endothelial damage, and hypercoagulability. The resulting model exhibits a substantial thrombotic burden involving multiple venous sinuses simultaneously. The induced thrombus remains stable for at least 1 week. The 2,3,5-triphenyltetrazolium chloride (TTC) staining demonstrates that this model can consistently produce large-area venous cerebral infarction, which persists for up to 7 days. This model can even induce epileptic seizures in rats, a capability that previous models were unable to achieve. The disruption of the blood-brain barrier was observed using Evans blue (EB) staining. Therefore, the severe CVST model established through semi-ligation combined with ferric chloride and thrombin administration more accurately replicates the pathophysiological progression of severe CVST in humans, demonstrating stability and reliability.

Introduction

Cerebral venous sinus thrombosis (CVST) is a distinct form of cerebrovascular disorder that differs from arterial stroke. It occurs more frequently in younger individuals^1,2, particularly in pregnant women³, and its incidence has been rising steadily in recent years. An epidemiological study conducted in Australia indicates that the annual incidence of CVST ranges from 13.0 to 15.7 cases per million individuals⁴. In the United States, the incidence of CVST exhibits an upward trend with age. Nevertheless, the peak age of onset for women is lower compared to that f....

Protocol

The experimental protocol was approved by the Animal Experiments and Experimental Animal Welfare Committee of Capital Medical University (AEEI-2020- 119) in Beijing, China, and was conducted in compliance with the institution's Animal Care and Use Committee regulations.

1. Animal preparation

1. Randomly assign healthy adult male Sprague-Dawley (SD) rats, with an average body weight of 300 ± 10 g, to either the experimental group or the sham operation group. All the animals were maintained on an alternating 12 h light/dark cycle with access to food and water ad libitum.

Results

Modeling process
Figure 1A depicts the SSS of the rat, as denoted by the arrow. The rostral and caudal of the SSS were semi-ligated (Figure 1B), followed by application of ferric chloride-soaked thread onto the surface of ligated regions (Figure 1C). Thrombin was subsequently injected into the ligated segments to induce thrombosis. The area denoted by the arrow, where thrombin was injected, has turned black (

Discussion

A novel rat model of severe CVST constructed by semi-ligation combined with ferric chloride and thrombin cannot only simulate blood stasis and endothelial injury, but also the hypercoagulable state of blood, that is, it simulates the three elements of thrombosis formation²⁵. What is even more exciting is that the rat model developed using this method is capable of exhibiting epileptic seizures. Epileptic seizures represent a notable clinical manifestation in patients with CVST, occurring in approx.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2,3,5-triphenyltetrazolium chloride	Sigma-Aldrich
40% ferric chloride	Tianjin Zhiyuan Chemical Reagent Co., Ltd., China
Evans blue	Sigma-Aldrich
Heating pad	Harvard Apparatus Holliston, MA, USA	50-7061-f,
High-speed dental drill	Saeshin, Busan, South Korea	Strong-207B
Laser speckle meter	PeriCam PSI System, Sweden
Microscope	Carl Zeiss, Inc., Berlin, Germany
Polyamide suture	Ningbo Chenghe Micro Apparatus Factory, China
Stereotaxic frame	David Kopf Instruments, Tujunga, California, USA
Thrombin	Chang Chun Lei Yunshang Pharmaceutical Co., Ltd., China