Establishment of a Rat Model of Superior Sagittal-Sinus Occlusion via a Thread-Embolism Method

Summary

Here, we establish a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis via a thread-embolization method, and the stability and reliability of the model were verified.

Abstract

The mechanisms contributing to the natural onset of cerebral venous sinus thrombosis (CVST) are mostly unknown, and a variety of uncontrollable factors are involved in the course of the disease, resulting in great limitations in clinical research. Therefore, the establishment of stable CVST animal models that can standardize a variety of uncontrollable confounding factors have helped to circumvent shortcomings in clinical research. In recent decades, a variety of CVST animal models have been constructed, but the results based on these models have been inconsistent and incomplete. Hence, in order to further explore the pathophysiological mechanisms of CVST, it is necessary to establish a novel and highly compatible animal model, which has important practical value and scientific significance for the diagnosis and treatment of CVST. In the present study, a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis was established via a thread-embolization method, and the stability and reliability of the model were verified. Additionally, we evaluated changes in cerebral venous blood flow in rats after the formation of CVST. Collectively, the SD-rat SSS-thrombosis model represents a novel CVST animal model that is easily established, minimizes trauma, yields good stability, and allows for accurately controlling ischemic timing and location.

Introduction

Cerebral venous sinus thrombosis (CVST) is a rare disease of the cerebral venous system that accounts for only 0.5-1.0% of all causes of stroke but has a relatively high occurrence rate in children and young adults¹. During autopsy, CVST was found to be the cause of 10% of cerebrovascular disease deaths². Thrombosis can occur in any part of the intracranial venous system. The superior sagittal sinus (SSS) is one of the most commonly affected areas in CVST and can involve multiple blood vessels. Owing to stenosis or occlusion of the venous sinuses, intracranial venous return is blocked, which is often accompanied by increased intracranial pressure³. The clinical manifestations of CVST are complex and vary over time; although there is a lack of specificity of symptoms, the most common symptoms include headache (77.2%), seizures (42.7%), and neurological deficits (39.9%). In severe cases, coma and even death may occur^4,5. In recent years, due to the overall improvement of medical and health standards and public health awareness, the proportion of related risk factors has changed, the proportion of trauma and infection has decreased, and the proportion of CVST caused by pregnancy, puerperium, oral contraceptives, and other reasons has gradually increased⁵.

At present, the pathogenesis of CVST is still not well understood. To explore CVST in depth, further pathophysiological research is needed. However, most of these research methods are invasive and therefore difficult to implement clinically. Owing to many limitations of clinical research, animal models have irreplaceable advantages in terms of basic and translational research.

The cause of CVST is complex, as its initial onset is often unrecognized and the location of thrombus formation is highly variable. Fortunately, animal models can achieve better control of these factors. In the past few decades, a variety of CVST animal models have been established, and each model has its own disadvantages. According to different production methods, they can be roughly divided into the following categories: the simple SSS-ligation model^6,7; the SSS internal-injection-accelerator model⁸; the ferric-chloride-induced SSS thrombosis model⁹; the photochemical-induced SSS thrombosis model¹⁰; and the self-made embolism-occlusion SSS model¹¹. However, most of these models are unable to circumvent invasive damage to the animal's cerebral cortex and are not able to accurately control the ischemic time and location. In some models, the thrombus will recanalize spontaneously; in other models, the SSS becomes permanently occluded. In addition, complicated operations and/or serious injuries may affect subsequent pathophysiological findings in these models.

In the present study, a thread plug was inserted into the SSS of Sprague-Dawley (SD) rats to successfully establish a CVST model that minimized damage, enabled precise controllability, and yielded good stability. Additionally, small-animal magnetic resonance imaging (MRI) and laser-speckle blood-flow imaging were combined to verify the model's effectiveness. We evaluated changes in cerebral blood flow before and after establishment of our model, as well as evaluated the stability of our model, laying a foundation for further studies exploring the occurrence, development, and related pathophysiological mechanisms of CVST.

Protocol

Procedures involving animal subjects have been approved by the Medical Norms and Ethics Committee of Wenzhou Medical University and are in accordance with the China legislation on the use and care of laboratory animals.

1. Preparation of the thread plug, SD rats, and experimental equipment

1. Use a nylon thread with a diameter of 0.28 mm as the main body of the thread plug.
   NOTE: The softness and hardness of the nylon thread should be moderate.
2. Cover one end of the nylon thread with silicone material. The length of the silicone part of the thread plug is approximately 1.2 cm, and the diameter is approximately 1.2 mm. The head end is tapered, and the silicone part is cylindrical. Reserve another 5-7 cm. The nylon thread is easy to clamp and can be cut according to specific needs after the operation.
3. Use 75% ethanol to soak the thread plug for 3 min before the operation and rinse any residual ethanol with normal saline before plugging.
4. Select 12 male SD rats weighing between 280 and 320 g, and randomly divide them into a sham group and experimental group (n = 6 per group). After a week of environmental adaptation, fast the rats for 12 h and water-deprive them for 4 h before the operation.
5. Prepare the following experimental equipment needed for the experiment: a small animal anesthesia machine, a brain stereotaxic instrument, a dissecting microscope, a high-speed skull drill, scissors, tweezers, vascular forceps, a needle holder, a needle thread, a 2 mL syringe, a laser-speckle blood-flow imaging system, and a small-animal MRI scanner.

2. Construction of SD-Rat SSS-Embolization Model via Thread Embolization

1. Place the SD rat into an anesthesia-induction box and use a small-animal anesthetic machine to deliver 4% isoflurane to induce anesthesia. Thereafter, use forceps to confirm that the SD rat's hind limbs and toes do not respond to moderate pinching.
2. Quickly fix the SD ratwith shaved top hair in the prone position on a brain stereotaxic device. Maintain anesthesia with 1.5-2.0% isoflurane (at a speed of 0.5 L/min), and stabilize the respiratory rate at 40-60 breaths/min. Stabilize the rat's body temperature via a heating pad at 37±0.2 °C.
   1. Apply sterile ophthalmic eye lubricant after the rat is placed on the stereotaxic frame to protect the corneas from drying during anesthesia.
3. Sterilize the surface on the top of the rat's head with 5% povidone iodine alternating three times with 75% ethanol. Make a skin incision (2.0-cm long) in the middle of the head, and then carefully peel off the top fascia and periosteum to fully expose the skull.
   1. Confirm the positions of the anterior fontanelle, posterior fontanelle, coronal suture, sagittal suture, and herringbone suture.
4. Use the area between the coronal suture and the herringbone suture as the blood-flow observational area. To prevent the skull from affecting observation during laser-speckle blood-flow imaging, thin the skull in the observation area until the blood vessels are clearly visible. The size of the thinned skull should be approximately 1.0 cm × 1.0 cm. This step and the following are all performed under a dissecting microscope.
   1. During skull grinding, use normal-temperature saline to rinse the drill repeatedly to avoid high-temperature burns to the cerebral cortex.
5. Use a high-speed drill to grind the skull within a 6.0 mm x 4.0 mm bone window centered at bregma to expose the SSS of the bregma area.
   1. Use normal saline to cool the skull during grinding. When the skull becomes thin, use tweezers to carefully remove the remaining bone pieces to avoid tearing the SSS.
6. Choose a suitable thread plug, use the SSS bregma point as the plug point, carefully puncture it with a 2 mL syringe needle, and quickly insert the thread plug head into the plug point.
   1. At this time, the angle between the end of the thread-plug head and the SSS should be approximately 30-45°; then adjust the angle between the end of the thread plug and the SSS to 0-10 degrees, and slowly insert the SSS into the center until the head reaches the posterior edge of the sinus confluence. Thereafter, cut off the excess part of the tail.
      ​NOTE: Rapid bleeding may occur when the SSS is punctured. If the end of the thread plug cannot be quickly inserted into the plug point at one time, use a small gauze or cotton ball to gently press the plug point while slowly sliding down to expose the plug point carefully, and then quickly insert the end of the wire bolt into the SSS. After the thread plug is inserted, if there is bleeding at the plug point, hemostatic materials such as a gelatin sponge can be used to stop the bleeding.

3. Detection of Blood Flow on the Brain Surface of SD Rats

1. Use a laser light source to uniformly illuminate the blood-flow observational area. The reflected light is collected by a camera and is transmitted to a computer for analysis. Use the following parameter settings for the laser-speckle blood-flow imaging system: wavelength: λ = 785 nm; and image exposure time: T = 10 ms.
2. Center the SD-rat blood-flow observational area into the field of view of the laser-speckle blood-flow imaging system and conduct continuous monitoring of blood flow on the brain surface for 2 min. Collect and process the blood flow data before and after embolization for each SD rat, and obtain the laser-speckle blood-flow map of the observed area.
3. Repeatedly rinse the operated area with normal saline to wash away bone debris and residue. Suture the skin (0# thread) and disinfect with iodophor.
4. Maintain body temperature until the rat awakens after surgery and then house in a single cage with food and water provided ad libitum. The sham group cannot be plugged.
5. After data collection is completed, perform post-processing.
   1. Complete selection of the region of interest (ROI) via the tools provided by the laser-speckle blood-flow imaging system software. The values obtained are the average blood-flow value in the ROI, and the local cerebral-blood-flow values before and after embolization. Use the blood-flow value before embolization as the baseline value.
   2. Select four ROIs and measure the relative change in cerebral blood flow in each ROI, expressed as the percent change from the baseline value.

4. Detection of thread position on small animals MRI

1. Use the following T2-weighted imaging (T₂WI) parameters for MRI imaging system: time of echo (TE) = 33 ms, time of repeat (TR) = 3000 ms, number of excitation (NEX) = 4, Slices = 28, slice thickness = 0.8 mm, matrix size = 256*256 mm², flip angle = 80°, field of view (FOV) = 30*30 mm², scan time = 6 min 24 s; magnetic resonance angiography (MRA) parameters were set as follows: TE = 4.4 ms, TR = 12 ms, NEX = 4, slices = 80, slice thickness = 0.4 mm, matrix size = 256*256 mm², flip angle = 80°, FOV = 30*30 mm², scan time = 16 min 23 sec 40 ms.
2. Fix the animal on the MRI scanning table, calibrate the brain position by positioning scanning, and perform T₂WI and MRA sequence scanning after confirming the position.
3. Use continuous anesthesia via the animal anesthesia machine during the detection. Then, euthanize the SD rats by intraperitoneal injection of excessive pentobarbital.
4. Image acquisition and post-processing: after collecting the image data, in order to observe the state of the thread plug in the SSS more clearly, use the pseudo color enhancement method to display the T₂WI image of the rat brain.

Results

To establish the SD-rat SSS-thrombosis model via the suture method, the suture should be prepared in advance (Figure 1A), and the equipment required for the experiment (Figure 1B) should be prepared. Due to the delicate nature of the operation, the preparation of the model needs to be completed under a dissecting microscope. The main steps are shown in Figure 2. To facilitate the description of the specific details of the blood-flow...

Discussion

In this study, a new type of CVST model was successfully established by inserting a self-made thread plug into the SSS of SD rats. Additionally, laser-speckle blood-flow imaging and small-animal MRI were combined to monitor changes in blood flow on the brain surface of SD rats before and after the embolization in order to standardize ischemic timing and location.

In 1989, Longa et al. made a reversible MCA occlusion model by retrogradely inserting a self-made nylon suture into the external car...

Materials

Name	Company	Catalog Number	Comments
2 mL syringe	Becton,Dickinson and Company	301940
brain stereotaxic instrument	Shenzhen RWD Life Technology Co., Ltd	68025
dissecting microscope	Wuhan SIM Opto-technology Co.	SIM BFI-HR PRO
high-speed skull drill	Shenzhen RWD Life Technology Co., Ltd	78046
laser-speckle blood-flow imaging system	Wuhan SIM Opto-technology Co.	SIM BFI-HR PRO
needle holder	Shenzhen RWD Life Technology Co., Ltd	F31022-12
needle thread	Shenzhen RWD Life Technology Co., Ltd	F33303-08
scissors	Shenzhen RWD Life Technology Co., Ltd	S13029-14
silica gel	Heraeus Kulzer	302785
small animal anesthesia machine	Shenzhen RWD Life Technology Co., Ltd	R540
small-animal MRI	Bruker Medical GmbH	Biospec 94/30 USR
tweezers	Shenzhen RWD Life Technology Co., Ltd	F11029-11
vascular forceps	Shenzhen RWD Life Technology Co., Ltd	F22003-09