Photothrombosis Combined With Thrombin Injection Establishes A Rat Model Of Cerebral Venous Sinus Thrombosis

Backgroup:Cerebral venous sinus thrombosis (CVST) is a rare but life-threatening disease with multifactorial etiology and highly variable clinical symptoms. Due to thrombosis of the cortical veins and major sinuses, brain edema and venous infarction as well as intracranial hypertension frequently occur in patients with CVST, resulting in deep coma, hemorrhage, deficits in neurological function, and even death. The estimated annual incidence is 3 to 4 cases per 1 million population and up to 7 cases per 1 million among children. However, the morbidity of CVST increased year by year that accounting for 1% of all strokes. Its epidemic factor are mostly oral contraceptives, pregnancy, postpartum, and about 75 percent of the adult patients are women. During the past decade, increased awareness of the diagnosis, improved neuroimaging techniques, and more effective treatment have improved the prognosis. More than 80 percent of all patients now have a good neurologic outcome.Recently, animal models have elucidated pathophysiologies such as edema formation, disruption of the blood-brain barrier (BBB) and hemodynamical alterations which allow evaluation of new therapies for CVST. However, the variety of methods of establishing CVST animal models results in great difference in the previous researches. An in-depth understanding of CVST requires an animal model that has the following three critical features:(i) reproduces the pathophysiology observed in humans with CSVT; (ii) follows the pattern of thrombosis and pathology observed in humans; and (iii) provides a platform for the assessment and development of novel therapeutic approaches.Previous studies have utilized various techniques to establish animal models, including superior sagittal sinus (SSS) ligation, SSS ligation combined with injecting of thrombogenic substances, application of FeCl3, vascular interventional embolization, as well as photochemical thrombosis. Previous studies have confirmed that cerebral venous infarction will develop in the brain parenchyma if the draining cortical veins are occluded. The more cortical veins and bridge veins involved in thrombosis the more serious damage in cerebral cortex once the collateral circulation was occluded. Unfortunately, it is difficult to find an animal model that involves thrombosis of both sinuses and veins without permanent ligation of the SSS.Photosensitizer that circulating in the cerebrovascular produce photochemical reactions when irradiated by its excitation light, thereby releasing oxygen free radicals. Oxygen free radicals are harmful metabolic substances, which can damage biofilm system and result in intracellular oxidative phosphorylation disorder. On the one hand, oxygen free radicals give rise to unsaturated fatty acid lipid peroxidation on vascular endothelial cell membrane. Increased lipid peroxides can damage the balance of PGI2 and TXB2, and promote adhesion of platelet aggregation, activation of coagulation process. On the other hand, free radicals can also damage the endothelial cells, and lead to detachment of endothelial cells from the blood vessel wall, exposing the underlying basement membrane endothelium. Subendothelial layer is rich in glycosaminoglycans and collagen material, which can promote platelet adhesion and aggregation. Leukocyte adhered to endothelial cells after endothelial cells damaged, which ultimately accelerated the formation of thrombus in the blood vessel when creating a vicious cycle.The photochemical formation of thrombus is similar to that of human cerebral thrombosis. In addition, irradiation or vascular photosensitizer itself makes no mechanical damage, which is similar to the clinical pathogenesis physiology of thrombotic disease. Thrombus was made up with red blood cells, platelets and fibrin when produced with photothrombosis, and the red thrombus is similar to that of human clinical pathology. The method may cause platelet aggregation and vascular endothelial cell injury, and provides possibility for the research of anti-platelet aggregation drugs and antithrombotic drugs. Therefore, photothrombossis can be widely used in basic and clinical research CVST other cerebrovascular disease. We utilized a method based on existing experimental models, in which photochemical thrombosis combined with thrombin injection created a clinically relevant cerebral sinus-vein model in the rat. This model may provide a better platform for in-depth research on the pathology of CVST and assessment of possible treatments.CVST is a rare, multi-venous etiology of ischemic stroke, rating 0.5% of stroke. However vein thrombosis in different positions, cortical bridging veins and venous involvement or not result in diverse clinical symptoms. It will be easily misdiagnosed for the reason of the lack of characteristic and no significant radiological features in acute phase. In recent years, with the development of diagnostic techniques and treatments, diagnosis rate of venous sinus thrombosis increased, and most patients were treated early, which leads to good prognosis and low mortality rate. However, the in-depth study of pathogenesis and physiological pathology of CVST still needs further exploration. Pathophysiological changes CVST is a complex process, the dynamic changes in the development process of judging the prognosis of the disease is particularly important, however, few studies were reported at home and abroad.In this experiment, we employed a sinus-vein thrombosis model in the rat to evaluate the effect of CVST on BBB permeability, brain edema, brain tissue infarction, as well as neurological function over 7 days. Our results suggest an optimal period of clinical intervention for patients afflicted with CVST, which will facilitate clinical treatment and prognosis.Part I Photothrombosis combined with thrombin injection establishes a rat model of cerebral venous sinus thrombosisObjective:This part is to develop a novel CVST rat model for studying clinically and experimental aspect and to detect the location and extension of thrombus. This will establish foundation for evaluating stability and reproducibility as well as exploring dynamic physiopathological changes of the model.Materials and methods:The Wistar rat was placed in a heat pad to keep body warm at 37℃, then venous cannulation was performed on the tail vein. After that, The animal was placed in the sphinx position and fixed within a stereotaxic frame. The skull was exposed by performing a 1.5-cm midline skin incision. H2O2 was then used to expose the skull. A high-speed dental drill monitored with a surgical microscope created a cranial window between the lambda and bregma. We avoided thermal injury to the cortex by continuously cooling the drill tip with cold saline. The SSS and bridge veins were gently exposed and care was taken to keep the dura intact. We then turned on a spot size-adjustable, stabilized green light Diode Pumped Solid State laser (DPSS) laser system that was affixed to the gripper of the stereotaxic frame. Green light at 532 nm was directed vertically from the laser device onto the SSS. Irradiation intensity was adjusted to 19.05 mw/mm2. Regulating knobs on the stereotaxic frame were used to position the green light spot on the surface of the SSS,0.5mm rostral to lambda or 0.5mm lambda to rostral. Beam diameter was adjusted to 1.0mm under control of the microscope to cover the surface of the SSS through an optical lens. Rose bengal solution was administered by intravenous injection into the tail vein at a rate of 1 mg/kg/min for 10min, until dark blood clots could be visualized in both the posterior and anterior SSS. Next, a syringe needle was inserted into the SSS rom the rostral to caudal direction. Thrombin was then slowly injected into the SSS for 10min, and the syringe needle was carefully withdrawn. Complete thrombosis was subsequently confirmed by fluorescence angiography.Results:Fifteen minutes after thrombin injection, a large black blood clot was visualized in the SSS under the surgery microscope. Fluorescence angiography showed that the black thrombus extend to SSS, bridge veins and cortical veins.Conclusions:Photochemistry produced free radical which injured endothelial cells and tore them off from the vessel lumen exposing the underlying basement membrane. The subendothelial layer was rich in glycosaminoglycans and collagen, which promoted platelet adhesion and aggregation, activation of the coagulation process in the rostral and lambda of SSS. Thrombin which spread into the bridging veinsand cortical veins, promoting coagulation of blood in the blood vessels. We confirmed the thrombosis in the superior sagittal sinus, bridging veins and cortical veins as to establish a rat model of sinus-veins thrombosis.Part II Explore the dynamic physiopathological changes of the rat CVST modelObjective:To establish rat sinus-veins thrombosis model and explore the BBB, brain water content, neurological function, infarct volumes, angiogenesis and gliocyte proliferation in the infarct area.Materials and methods:143 Wistar rats were randomly divided into 3 groups: a model group, a sham-operated group, and a normal control group. Then rats in the experimental and sham-operated groups were allocated to time point subgroups:day 1, day 2, and day 7. Once the rats were deeply anesthetized, we performed cardiac perfusion and fixation. The brains, dura mater and SSS were then removed. Evans Blue dye examination was performed to detect the permeability of BBB; dry-wet weight to detect the brain water content; rotarod test system to examine the neurological deficits; TTC, HE and MAP-2 staining to detect the infarct volumes and scope; Immunohistochemical staining to study angiogenesis and gliosis in the infarct area.Results:Compared with the normal and sham-operated groups, BBB permeability measured by extravasation of Evans Blue dye, neurological deficits and brain water content increased significantly on day 1. Brain edema peaked on day 2 and corresponded with an increase in BBB permeability compared with the sham-operated group at various time points. BBB permeability, neurological deficits and brain water content then decreased gradually from day 2 to day 7. BBB permeability-induced damage in the parenchyma of the parietal lobe near the SSS, typically recovered by day 7. TTC, HE and MAP-2 staining showed that compared with the sham-operated group, infarction in the model group was visible in the brain parenchyma on day 1 and was more extensive on day 2. Brain tissue damage typically recovered due to a decrease in infarction area on day 7 compared with that on day 2. Moreover, ischemic infarct and petechial hemorrhages as well as edema occurred regularly in the infarct area. Immunohistochemical staining showed that microvessels remained unchanged but astrocytes decreased significantly in the infarct area on day 1 and day 2. However, angiogenesis and gliosis in the infarct area are observed on day 7.Conclusions:The study confirmed that the novel rat CVST model established by Photothrombosis combined with thrombin injection reproduces the pathophysiology observed in humans with CS VT. Our results that studying clinically relevant aspects of CVST and investigating its dynamic pathophysiological changes during a 7-day period indicate a period of optimal clinical intervention for patients with CVST, which may reduce the probability of dependency and death.