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A New Thrombosis Model of the Superior Sagittal Sinus Involving Cortical Veins
Peer-Review Reports
A New Thrombosis Model of the Superior Sagittal Sinus Involving Cortical Veins Guangwen Li1, Xianwei Zeng4, Tailing Ji4, Vance Fredrickson5, Tony Wang5, Mohammed Hussain5, Changhong Ren2, Jian Chen3, Chaitanya Sikhram5, Yuchuan Ding5, Xunming Ji3
Key words Cortical vein - Hemorrhage - Infarction - Superior sagittal sinus - Thrombosis
- OBJECTIVE:
Abbreviations and Acronyms CVST: Cerebral venous sinus thrombosis DWI: Diffusion-weighted image FOV: Field-of-view MRI: Magnetic resonance imaging MRV: Magnetic resonance venography SSS: Superior sagittal sinus TE: Echo time TR: Repetition time TTC: Triphenyltetrazolium chloride WW: Wet weight
- METHODS:
-
From the 1Cerebrovascular Diseases Research Institute, 2Institute of Hypoxia Medicine, and 3Department of Neurosurgery, Xuanwu Hospital, Capital Medical University, Beijing, People's Republic of China; 4Department of Neurosurgery, Affiliated Hospital of Weifang Medical College, Weifang, People's Republic of China; and 5Department of Neurological Surgery, Wayne State University School of Medicine, Detroit, Michigan, USA To whom correspondence should be addressed: Xunming Ji, M.D., Ph.D. [E-mail: [email protected]] Citation: World Neurosurg. (2013). http://dx.doi.org/10.1016/j.wneu.2012.11.062 Journal homepage: www.WORLDNEUROSURGERY.org
Patients with cerebral sinus and cortical venous thrombosis develop venous infarcts in approximately 50% of cases, resulting in serious clinical symptoms. An animal model is needed to further clarify the underlying mechanisms and consequences surrounding cerebral venous sinus thrombosis, particularly for severe ones. Adult male Sprague-Dawley rats were used to develop a new superior sagittal sinus thrombosis model involving cortical veins. The superior sagittal sinus was exposed and ligated. A microcatheter was inserted into the sinus, then both common carotid arteries were temporary occluded to reduce cerebral blood flow, and thrombin was injected into the sinus. Twenty-four hours later, after evaluating neurological function and obtaining a magnetic resonance imaging, animals were sacrificed and data pertaining to brain water content, infarct volume, and tissue histology was collected.
- RESULTS:
Superior sagittal sinus thrombosis and brain infarction were detected in all rats (100%). Hemorrhagic infarction, when present, and brain edema were observed in the brain parenchyma of the parietal lobe. The rate of hemorrhage was 59%, which is similar to that seen clinically in patients with superior sagittal sinus thrombosis. Brain edema, as measured by brain water content percentage, was significantly increased in thrombosed animals compared with sham-operated animals (80.8% 0.55% vs. 78.8% 0.14%, P < 0.05). Infarct volumes were 53.02 7.91 mm3.
- CONCLUSIONS:
We suggest that our modified model of superior sagittal sinus thrombosis, involving cortical veins, is suitable for the study of its underlying mechanisms, as well as therapeutic approaches directed at the disease.
Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2013 Elsevier Inc. All rights reserved.
INTRODUCTION Cerebral venous sinus thrombosis (CVST) is a rare type of cerebrovascular event that affects three to four people per million, and it accounts for less than 1% of all strokes (3, 20). Its onset is often without warning, and its clinical presentation is highly variable. Isolated dural sinus thrombosis without involvement of the cerebral cortical veins results in the clinical syndrome of intracranial hypertension, with papilledema, headache, and vomiting (21). Patients with cerebral cortical venous thrombosis develop venous infarcts in about 50% of cases, often resulting in serious clinical symptoms, such as seizure (4), focal
neurological deficits, and loss of consciousness (17). Currently, the pathogenesis of cranial sinus thrombosis remains poorly understood, and effective treatment strategies have yet to be elucidated. As such, the construction of a rat model that includes both cerebral sinus and cortical venous thrombosis, to study pathogenesis and therapeutics, is important to advance future treatment strategies for CVST victims. The superior sagittal sinus (SSS) is the most common location of cerebral venous thrombosis in humans (4); as a result, many models of SSS thrombosis have been developed. Such models include ligation of the sinus (10, 11), injection of thrombogenic
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substances (22), balloon occlusion (15, 23), occlusion by photothrombosis (7), and application of filter paper soaked in 40% ferric chloride (18, 19). These models produced a thrombus confined to the SSS, with no thrombus formation in the cortical veins. Previous studies have shown that a thrombus, when restricted to the SSS, will not affect cerebral blood flow, nor will it lead to venous infarction and hemorrhage (10, 14), sequelae that are often seen in human instances of cerebral venous thrombosis. Recently, Srivastava et al. (18) showed that ferric chloride produces a large infarction, but this could be attributed to the inherent caustic properties of the ferric chloride itself. Thus, these animal models of SSS
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thrombosis represent less than ideal models of cerebral venous thrombosis in humans. In the present study, we modified the thrombosis model of the superior sagittal sinus involving cortical veins of H. Nakase et al. (11), hence producing a thrombosis model similar to that seen clinically. In the present model, we injected thrombin into the SSS together with the cortical veins, which led to cerebral edema and often hemorrhagic infarction, producing effects similar to those seen in patients with severe CVST. This model may provide a new means of studying the pathogenesis, as well as treatment therapies, for CVST. METHODS Animal Preparation Male Sprague-Dawley rats (280e310 g; Beijing Vitalriver Experimental Animal Co., Beijing, China) were studied. All procedures were conducted according to institutional guidelines and were in compliance with regulations formulated by the Animal Care and Use Committee, Capital Medical University of Beijing, China. A total of 49 rats were used in the experiment, and they were randomly divided into two groups: a study group (n ¼ 32) in which thrombi were induced in the SSS and cortical veins, and a sham-operated group (n ¼ 17). Five rats died from intracranial hemorrhage in the study group during/following the procedure, giving a study group mortality rate of 15.6%. A total of 44 rats survived; of the surviving rats, both groups were further divided into three groups and were used in statistical analysis. Rats allocated to subgroups were used for 1) edema studies (n ¼ 11 for study group; n ¼ 11 for sham group); 2) 2,3,5-triphenyltetrazolium chloride (TTC) staining (n ¼ 11 for study group; n ¼ 3 for sham group); 3) histologic examination (n ¼ 5 for study group; n ¼ 3 for sham group); and the data were analyzed 24 hours after induction of the thrombus. The animals were maintained on an alternating 12-hour light/dark cycle with free access to food and water. Surgical Preparation Anesthesia was induced by 5% isoflurane, and, throughout the surgery was maintained with 2%e3% isoflurane delivered in a mixture of 70% nitrous oxide and 30%
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A NEW THROMBOSIS MODEL
oxygen by a face mask. During anesthesia, body temperature was monitored with a rectal probe and maintained at 37 C 0.5 C using a thermostatically controlled heating pad (Harvard Apparatus 50-7061-f, Holliston, MA, USA). In addition, PE-50 tubing was inserted into the left femoral artery for monitoring mean arterial blood pressures and obtaining blood samples to determined blood gases and glucose during the operation. Blood samples were obtained immediately before and at 30 minutes after the craniotomy, and at the beginning of SSS occlusion. The surgical procedure was performed as follows: a midline neck incision was made, the common carotid arteries were exposed bilaterally, and a suture was placed under the artery on each side. Next, the animal was placed in the sphinx position with the head fixed at midline using a stereotaxic frame (David Kopf Instruments, Tujunga, California, USA). A 2.0-cm midline skin incision was performed, exposing the calvaria. A longitudinal cranial window (8 4 mm), centered on the coronal and lambda sutures, exposing the sinus and bilateral parasagittal cortex, was created using a dental high-speed drill (Strong207B, Saeshin, Busan, South Korea) under microscopic control (Carl Zeiss, Inc., Berlin, Germany). The drill-tip was cooled continuously with physiologic saline to avoid thermal injury to the underlying cortex. Care was taken to avoid lesions of the SSS and bridging veins, and, after the window formation procedure, the dura mater was completely intact. The SSS was exposed and permanently ligated rostrally and caudally with 8-0 Prolene suture (Ethicon Endo-Surgery, Inc., Cincinnati, OH, USA). A microcatheter-shaped PE-50 (diameter, 0.35e0.38 mm) was inserted into the SSS rostral to the caudal ligation and visualized using an operating microscope (Nikon 80i, Nikon Instruments Inc., Melville, NY, USA). Then the microcatheter was sutured to the SSS to avoid leakage of the thrombin during injection. Both common carotid arteries were then temporarily occluded with suture; next, thrombin (100 mL, 50 IU/mL; ChangChun Grand Sinlo Pharmaceutical Co., Ltd., China) was injected into the SSS during 1 minute, by hand. The extended craniotomy, along the length of the SSS, allowed for visualization of both the thrombin injection and the induced thrombus
formation, providing assurance of maximal thrombus extension into the cortical veins. After the thrombin injection, the sutures were removed from the carotids to allow for reperfusion. The total carotid occlusion time was 1 minute. The microcatheter was withdrawn after a dark, solid blood clot was visible in the SSS, occurring approximately 5 minutes after thrombin injection. Upon completion of the procedure, the field was flushed with physiologic saline and the incision was closed. Animals were then returned to their respective cages and allowed to recuperate from anesthesia. Animals in the sham-operated group underwent anesthesia, had their femoral arteries cannulated, underwent surgery to form the longitudinal cranial window, and then the incision was closed. Neurological Evaluation Neurological evaluations were done in a blinded manner. The rats were subjected to a neurological evaluation before the surgery, as well as 0.5 and 24 hours after the surgery. Animals were scored as follows: 0 ¼ no observable neurological deficit (normal); 1 ¼ failure to extend left forepaw on lifting the whole body by tail (mild); 2 ¼ circling to the contralateral side (moderate); 3 ¼ leaning to the contralateral side at rest or no spontaneous motor activity (severe) (19). Magnetic Resonance Imaging Protocol The brains were observed for evidence of infarction and thrombus by magnetic resonance imaging (MRI) 24 hours after surgery. Animals were reanesthetized by an intraperitoneal injection of chloral hydrate (36 mg/100 g body mass) and placed in the prone position, with their heads inside birdcage radiofrequency coins (Siemens, Deerfield, IL, USA). The MRI imaging was performed using a 3.0-T Magnetom Verio syngo (Siemens). T1-weighted images, T2weighted images, diffusion-weighted images (DWI), and magnetic resonance venography (MRV) were acquired. (Tlweighted ¼ repetition time [TR] 500 ms, echo time [TE] 13 ms, field-of-view [FOV] 78 mm, 2.0-mm slice thickness, 11 slices; T2-weighted ¼ TR 4000 ms, TE 92 ms, FOV 64 mm, 2.0-mm slice thickness, 11 slices; DWI ¼ TR 3500 ms, TE 80 ms, FOV 94 mm, 2.0-mm slice thickness, 11 slices; MRV ¼ TR 46 ms, TE 5.14 ms, FOV 120 mm, 2.0-mm slice thickness, 30 slices.)
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Brain Water Content Twenty-four hours after CVST, the rats were sacrificed with chloral hydrate (40 mg/100 g body mass) administration, and the brains were subsequently removed. The cerebellum, pons, and olfactory bulbs were then removed from each brain and each cerebrum was weighed to determine its wet weight (WW). Subsequently, the brains were placed into a 110 C thermal oven for 24 hours, and then weighed to determine their dry weight. The formula (WW - Dry weight)/ WW 100% was used to calculate the water content percentage of each brain (6). 2,3,5-Triphenyltetrazolium Chloride Staining Rats were sacrificed with chloral hydrate (40 mg/100 g body mass) administration 24 hours after the surgery. Brains were harvested and cut into 2-mm thick coronal slices for TTC (Sigma-Aldrich, St. Louis, MO, USA) treatment. Brain slices were incubated in 1% TTC solution at 37 C for 20 minutes. Stained slices were fixed in 10% formalin solution. The decolorized area in each brain slice was determined using an image analysis program (Image J 1.44p; National Institutes of Health, Bethesda, Maryland, USA). The brain lesion volume was calculated by the product of average slice thickness (2 mm) and the sum of infarction area in all the six slices. The results were expressed as mean standard deviation. Histologic Examination Rats were sacrificed with chloral hydrate (40 mg/100 g body mass) administration, and then perfused cardiovascularly with physiologic saline and 4% paraformaldehyde in saline. The SSS and brains were collected. After paraffin embedding, 5-mm-thick coronal sections were cut and stained with hematoxylin-eosin (Sigma). The brains were viewed using a histologic light microscope (Nikon 80i), and observations were made regarding changes in the parenchymal tissue and SSS. Three sham-operated rats were used as controls for normal brain tissue and endothelial cells. Statistical Analysis Data were expressed as means standard deviation, and statistical analysis was performed with one-way analysis of variance and the t-test using SPSS for Windows,
A NEW THROMBOSIS MODEL
Table 1. Physiologic Parameters
Time
Mean Arterial Blood Pressure (mm Hg)
SaO2
pO2 (mm Hg)
pCO2 (mm Hg)
pH
Glucose (mg/dL)
Before sinus occlusion
91.7 4.9
98.6 1.03 116.66 7.66 46.78 3.00 7.42 0.06 132.3 10.6
Five minutes after occlusion
92.5 6.0
98.0 0.63 115.17 8.93 48.25 3.71 7.33 0.04 132.0 11.2
Values are expressed as mean SD. No significance was detected by analysis of variance analysis. pCO2, carbon dioxide partial pressure; pO2, oxygen partial pressure; SaO2, arterial oxygen percent saturation.
version 11.5 (SPSS Inc., Chicago, IL, USA). Statistical significance was set to P < 0.05. RESULTS Physiologic Variables In the study group, no significant differences were observed in the physiologic parameters (rectal temperature, blood alveolar oxygen tension, alveolar carbon dioxide tension, and pH, mean arterial blood pressure, glucose) as measured before and after venous occlusion (P < 0.05). Likewise, there were no significant differences in these variables when comparing the study group to the sham-operated control group (P < 0.05). Details regarding physiologic data are given in Table 1. Neurological Evaluation All animals exhibited a normal postural reflex (score ¼ 0) before surgery. Neurological scores in the study group were 1.74 0.59 at 0.5 hours after surgery, and 2.11 0.58 at 24 hours after surgery. There was no significant difference in neurological deficit at 0.5 and 24 hours after surgery. The study group displayed significant (P < 0.001) neurological deficits when compared with the sham-operated control group. The control group did not exhibit obvious neurological deficits (Figure 1). MRI Evaluation MRI confirmed that SSS occlusion and brain infarction were present in all animals, with hemorrhage occurring in 59% (16/27) of the animals. Figure 2 shows typical examples of the MRI findings. The MRV confirmed occlusion of the SSS after the operation (image E). The DWI image indicates the local brain infarction (image F).
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T1 and T2-weighted images indicate the presence of hemorrhagic infarction and brain edema, which contributed to a midline shift (images G and H). No brain lesions were observed in the images of sham-operated control rats (images AeD). Brain Water Content The brain water content of animals in the study group was 80.8% 0.55%, and in the sham-operated group was 78.8% 0.14%. There was a significant (P < 0.05) difference between the two groups (Figure 3). TTC Staining An SSS thrombus was detected in each of the study group animals, and infarctions were confirmed by TTC staining. Staining of the brain slices revealed a bilateral infarction in three animals and a unilateral infarction in eight animals. The brain lesion volume was 53.02 7.91 mm3 at 24 hours after the operation (Figure 4). No brain lesions were observed in the control group (not shown).
Figure 1. Time course of neurological deficit scores. In the study group, there was no significant difference between the neurological deficit scores at 0.5 and 24 hours after surgery. In comparison to the sham-operated control group, the study group displayed significant (*P < 0.001) neurological deficits. Sham-operated group, n ¼ 17; study group, n ¼ 27.
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Figure 3. The brain water content percentage in the sham-operated and study groups. There was a significant (*P < 0.05) difference observed between the two groups. Superior sagittal sinus thrombosus (SSST) indicates the study group animals with superior sagittal sinus thrombosis. Sham-operated group, n ¼ 11; study group, n ¼ 11.
The SSS was filled with blood, thrombus, and inflammatory cells determined by hematoxylin-eosin staining (Figure 5). DISCUSSION Several previous models of SSS have been developed (7, 10, 11, 15, 18, 19, 22, 23); however, in these models, the induced thrombus was confined to the SSS, with no thrombus formation in the cortical veins. Previous studies have shown that a thrombus, when restricted to the SSS, does not significantly alter cerebral blood flow, nor does it cause venous infarction and hemorrhage (10, 14). The present study demonstrates that an injection of thrombin
Figure 2. Magnetic resonance imaging (MRI) took place at the 24-hour time point. A magnetic resonance venography (MRV) image showing the superior sagittal sinus (SSS) in the normal shamoperated control rats (A, arrow). The arrow in E indicates filling defects at 24 hours after the operation. The MRV reveals the absence of a signal in the SSS and a normal flow signal in the anterior part of the SSS. Diffusion-weighted MRI (DWI) displays the hyperintense lesion (F, arrow). T1weighted MRI and T2-weighted MRI (G, H) show the infarction. In addition, the T2-MRI shows brain edema at 24 hours after the operation, especially in the left parieto-occipital lobes. Images from sham-operated control rats (AeD) show that no brain lesions were present. Superior sagittal sinus thrombosus (SSST) indicates the study group animals with superior sagittal sinus thrombosis.
Histologic Findings Hemorrhagic infarction, when present, and brain edema were both observed in the
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parenchyma of the parietal lobe. The image in Figure 5 illustrates that the distance between each neuron increased as a result of edema.
Figure 4. Triphenyltetrazolium chloridestained brain slices from animals in the study group. The unstained areas represent the infarction. Animal A exhibited an infarction on one side of the midline, and Animal B exhibited a bilateral infarction.
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Figure 5. Histologic images showing normal superior sagittal sinus (SSS) and brain tissue, and the effects of the induced thrombus. (A) SSS of a typical sham-operated animal. The sinus was surrounded by endothelial cells and no blood or thrombus was observed within the sinus. (B) SSS of a typical study animal. The sinus was filled with blood, thrombus (arrowhead) and inflammatory cells (arrow). (C) A typical coronal section of
into the SSS and cortical veins of rats can reliably model human CVST injury patterns, including infarction, parenchyma hemorrhage, and cerebral edema. In our proposed method, venous thrombosis was achieved by a combination of sinus ligation and thrombin injection. Ligation of the SSS produces venous stasis, a hemodynamic condition that favors thrombin activity and venous thrombosis (5). Furthermore, our proposed method involves bilateral occlusion of the common carotid arteries. This approach can reduce blood flow in the cortex by more than 60% (1), resulting in dramatically reduced cerebral perfusion pressure, thus allowing the thrombin to enter the cortical veins more easily. In addition, the thrombus in this animal model extended progressively into the cortical veins, a pattern often seen clinically in patients with CVST. Venous sinus thrombosis leads to disordered blood flow (reflux)
the SSS from an animal in the study group; the thrombus was in the SSS (arrow) and cortical vein (arrowhead). (D) Normal brain tissue from an animal in the sham-operated group. (E, F) Animals in the study group. Typical brain parenchyma bleeding (arrowhead) and edema (arrow). The distance between each neuron was wider than normal due to the edema. Scale bar (A, B, C), 1.25 mm; scale bar (D, E, F), 0.25 mm.
and increased venous pressure. As a result, the intracranial pressure increases and perfusion pressure decreases, leading to reduced cerebral blood flow. Spontaneous intracranial hypotension and low cerebral spinal fluid pressure have been shown to further accentuate thrombin activity and have been associated with cerebral venous thrombosis (8, 9, 12, 13, 24). An advantage of our proposed method is the ease at which the investigator can assess thrombus formation. It can be directly visualized, using microscopy, as a darkening in color of the dural walls comprising the SSS. Furthermore, no active bleeding was observed after the PE50 was removed. A potential shortfall of the model is that injection of thrombin into the SSS likely produces only a local hypercoagulable state. In our model, the hypercoagulable state was transient and local, whereas systemic hemodynamics
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likely remained relatively unchanged. As such, when using our rodent model, the investigator needs to be aware of the local hypercoagulability produced and how it differs from the systemic hypercoagulability often seen clinically. Headache is the most common symptom of CVST and occurs in approximately 90% of patients (4). Headache in patients with CVST is often reflective of increased intradural sinus pressure, a condition associated with parenchymal and cerebral edema (21). Venous thrombosis is associated with venous infarction and increased venous leakage, leading to vasogenic edema (2). Furthermore, circulatory stasis can impair the sodium pump and other blood-brain barrier functions, leading to cytotoxic edema (16). Total venous occlusion can lead to complications such as venous rupture and hemorrhage (16). Our method, unlike previously described models of venous
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thrombosis (7, 10, 11, 15, 22, 23), can induce many of the major complications seen in CVST, including infarction, hemorrhage, and edema. As such, we propose that our method is a more reliable model for assessing CVST and its complications compared with previously suggested models. REFERENCES 1. Choy M, Ganesan V, Thomas DL, Thornton JS, Proctor E, King MD, van der Weerd L, Gadian DG, Lythgoe MF: The chronic vascular and haemodynamic response after permanent bilateral common carotid occlusion in newborn and adult rats. J Cereb Blood Flow Metab 26:1066-1075, 2006. 2. Ducreux D, Oppenheim C, Vandamme X, Dormont D, Samson Y, Rancurel G, Cosnard G, Marsault C: Diffusion-weighted imaging patterns of brain damage associated with cerebral venous thrombosis. AJNR Am J Neuroradiol 22:261-268, 2001. 3. Einhaupl K, Stam J, Bousser MG, de Bruijn SF, Ferro JM, Martinelli I, Masuhr F: EFNS guideline on the treatment of cerebral venous and sinus thrombosis in adult patients. Eur J Neurol 17: 1229-1235, 2010. 4. Ferro JM, Canhao P, Stam J, Bousser MG, Barinagarrementeria F: Prognosis of cerebral vein and dural sinus thrombosis: results of the International Study on Cerebral Vein and Dural Sinus Thrombosis (ISCVT). Stroke 35:664-670, 2004. 5. Hassouna HI: Blood stasis, thrombosis and fibrinolysis. Hematol Oncol Clin North Am 14: xvii-xxii, 2000. 6. Kawai N, Kawanishi M, Okauchi M, Nagao S: Effects of hypothermia on thrombin-induced brain edema formation. Brain Res 895:50-58, 2001. 7. Kimura R, Nakase H, Tamaki R, Sakaki T: Vascular endothelial growth factor antagonist reduces brain edema formation and venous infarction. Stroke 36:1259-1263, 2005.
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8. Lai PH, Li JY, Lo YK, Wu MT, Liang HL, Chen CK: A case of spontaneous intracranial hypotension complicated by isolated cortical vein thrombosis and cerebral venous infarction. Cephalalgia 27: 87-90, 2007. 9. Lan MY, Chang YY, Liu JS: Delayed cerebral venous thrombosis in a patient with spontaneous intracranial hypotension. Cephalalgia 27: 1176-1178, 2007. 10. Nakase H, Heimann A, Kempski O: Local cerebral blood flow in a rat cortical vein occlusion model. J Cereb Blood Flow Metab 16:720-728, 1996. 11. Nakase H, Takeshima T, Sakaki T, Heimann A, Kempski O: Superior sagittal sinus thrombosis: a clinical and experimental study. Skull Base Surg 8:169-174, 1998. 12. Nardone R, Caleri F, Golaszewski S, Ladurner G, Tezzon F, Bailey A, Trinka E, Zuccoli G: Subdural hematoma in a patient with spontaneous intracranial hypotension and cerebral venous thrombosis. Neurol Sci 31:669-672, 2010. 13. Richard S, Kremer S, Lacour JC, Vespignani H, Boyer P, Ducrocq X: Cerebral venous thrombosis caused by spontaneous intracranial hypotension: two cases. Eur J Neurol 14:1296-1298, 2007. 14. Rottger C, Bachmann G, Gerriets T, Kaps M, Kuchelmeister K, Schachenmayr W, Walberer M, Wessels T, Stolz E: A new model of reversible sinus sagittalis superior thrombosis in the rat: magnetic resonance imaging changes. Neurosurgery 57:573-580, 2005. 15. Sarwar M, Virapongse C, Carbo P: Experimental production of superior sagittal sinus thrombosis in the dog. AJNR Am J Neuroradiol 6:19-22, 1985. 16. Schaller B, Graf R: Cerebral venous infarction: the pathophysiological concept. Cerebrovasc Dis 18: 179-188, 2004.
developing an experimental model. J Neurosci Methods 161:220-222, 2007. 19. Srivastava AK, Kalita J, Dohare P, Ray M, Misra UK: Studies of free radical generation by neurons in a rat model of cerebral venous sinus thrombosis. Neurosci Lett 450:127-131, 2009. 20. Stam J: Thrombosis of the cerebral veins and sinuses. N Engl J Med 352:1791-1798, 2005. 21. Tsai FY, Wang AM, Matovich VB, Lavin M, Berberian B, Simonson TM, Yuh WT: MR staging of acute dural sinus thrombosis: correlation with venous pressure measurements and implications for treatment and prognosis. AJNR Am J Neuroradiol 16:1021-1029, 1995. 22. Wang J, Ji X, Ling F, Luo Y, He X, Guo M, Li S, Miao Z, Zhu F, Xuan Y: A new model of reversible superior sagittal sinus thrombosis in rats. Brain Res 1181:118-124, 2007. 23. Wang J, Tan HQ, Li MH, Sun XJ, Fu CM, Zhu YQ, Zhou B, Xu HW, Wang W, Xue B: Development of a new model of transvenous thrombosis in the pig superior sagittal sinus using thrombin injection and balloon occlusion. J Neuroradiol 37:109-115, 2010. 24. Wang YF, Fuh JL, Lirng JF, Chang FC, Wang SJ: Spontaneous intracranial hypotension with isolated cortical vein thrombosis and subarachnoid haemorrhage. Cephalalgia 27:1413-1417, 2007.
Conflict of interest statement: This study was supported by New Century Excellent Talents from Ministry of Education (no. NCET-08-0625), Health Bureau of High Level Talent project (no. 2009-3-61), Chinese National Science Foundation (no. 30870854), and Board of Education Science and Technology Innovation Platform of Beijing (PXM2011014226-07-000070). Guangwen Li and Xianwei Zeng contributed equally to this work as co-first authors. Received 16 May 2012; accepted 19 November 2012
17. Sebire G, Tabarki B, Saunders DE, Leroy I, Liesner R, Saint-Martin C, Husson B, Williams AN, Wade A, Kirkham FJ: Cerebral venous sinus thrombosis in children: risk factors, presentation, diagnosis and outcome. Brain 128:477-489, 2005. 18. Srivastava AK, Gupta RK, Haris M, Ray M, Kalita J, Misra UK: Cerebral venous sinus thrombosis:
Citation: World Neurosurg. (2013). http://dx.doi.org/10.1016/j.wneu.2012.11.062 Journal homepage: www.WORLDNEUROSURGERY.org Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2013 Elsevier Inc. All rights reserved.
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A New Thrombosis Model of the Superior Sagittal Sinus Involving Cortical Veins Guangwen Li1, Xianwei Zeng4, Tailing Ji4, Vance Fredrickson5, Tony Wang5, Mohammed Hussain5, Changhong Ren2, Jian Chen3, Chaitanya Sikhram5, Yuchuan Ding5, Xunming Ji3
Key words Cortical vein - Hemorrhage - Infarction - Superior sagittal sinus - Thrombosis
- OBJECTIVE:
Abbreviations and Acronyms CVST: Cerebral venous sinus thrombosis DWI: Diffusion-weighted image FOV: Field-of-view MRI: Magnetic resonance imaging MRV: Magnetic resonance venography SSS: Superior sagittal sinus TE: Echo time TR: Repetition time TTC: Triphenyltetrazolium chloride WW: Wet weight
- METHODS:
-
From the 1Cerebrovascular Diseases Research Institute, 2Institute of Hypoxia Medicine, and 3Department of Neurosurgery, Xuanwu Hospital, Capital Medical University, Beijing, People's Republic of China; 4Department of Neurosurgery, Affiliated Hospital of Weifang Medical College, Weifang, People's Republic of China; and 5Department of Neurological Surgery, Wayne State University School of Medicine, Detroit, Michigan, USA To whom correspondence should be addressed: Xunming Ji, M.D., Ph.D. [E-mail: [email protected]] Citation: World Neurosurg. (2013). http://dx.doi.org/10.1016/j.wneu.2012.11.062 Journal homepage: www.WORLDNEUROSURGERY.org
Patients with cerebral sinus and cortical venous thrombosis develop venous infarcts in approximately 50% of cases, resulting in serious clinical symptoms. An animal model is needed to further clarify the underlying mechanisms and consequences surrounding cerebral venous sinus thrombosis, particularly for severe ones. Adult male Sprague-Dawley rats were used to develop a new superior sagittal sinus thrombosis model involving cortical veins. The superior sagittal sinus was exposed and ligated. A microcatheter was inserted into the sinus, then both common carotid arteries were temporary occluded to reduce cerebral blood flow, and thrombin was injected into the sinus. Twenty-four hours later, after evaluating neurological function and obtaining a magnetic resonance imaging, animals were sacrificed and data pertaining to brain water content, infarct volume, and tissue histology was collected.
- RESULTS:
Superior sagittal sinus thrombosis and brain infarction were detected in all rats (100%). Hemorrhagic infarction, when present, and brain edema were observed in the brain parenchyma of the parietal lobe. The rate of hemorrhage was 59%, which is similar to that seen clinically in patients with superior sagittal sinus thrombosis. Brain edema, as measured by brain water content percentage, was significantly increased in thrombosed animals compared with sham-operated animals (80.8% 0.55% vs. 78.8% 0.14%, P < 0.05). Infarct volumes were 53.02 7.91 mm3.
- CONCLUSIONS:
We suggest that our modified model of superior sagittal sinus thrombosis, involving cortical veins, is suitable for the study of its underlying mechanisms, as well as therapeutic approaches directed at the disease.
Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2013 Elsevier Inc. All rights reserved.
INTRODUCTION Cerebral venous sinus thrombosis (CVST) is a rare type of cerebrovascular event that affects three to four people per million, and it accounts for less than 1% of all strokes (3, 20). Its onset is often without warning, and its clinical presentation is highly variable. Isolated dural sinus thrombosis without involvement of the cerebral cortical veins results in the clinical syndrome of intracranial hypertension, with papilledema, headache, and vomiting (21). Patients with cerebral cortical venous thrombosis develop venous infarcts in about 50% of cases, often resulting in serious clinical symptoms, such as seizure (4), focal
neurological deficits, and loss of consciousness (17). Currently, the pathogenesis of cranial sinus thrombosis remains poorly understood, and effective treatment strategies have yet to be elucidated. As such, the construction of a rat model that includes both cerebral sinus and cortical venous thrombosis, to study pathogenesis and therapeutics, is important to advance future treatment strategies for CVST victims. The superior sagittal sinus (SSS) is the most common location of cerebral venous thrombosis in humans (4); as a result, many models of SSS thrombosis have been developed. Such models include ligation of the sinus (10, 11), injection of thrombogenic
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substances (22), balloon occlusion (15, 23), occlusion by photothrombosis (7), and application of filter paper soaked in 40% ferric chloride (18, 19). These models produced a thrombus confined to the SSS, with no thrombus formation in the cortical veins. Previous studies have shown that a thrombus, when restricted to the SSS, will not affect cerebral blood flow, nor will it lead to venous infarction and hemorrhage (10, 14), sequelae that are often seen in human instances of cerebral venous thrombosis. Recently, Srivastava et al. (18) showed that ferric chloride produces a large infarction, but this could be attributed to the inherent caustic properties of the ferric chloride itself. Thus, these animal models of SSS
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thrombosis represent less than ideal models of cerebral venous thrombosis in humans. In the present study, we modified the thrombosis model of the superior sagittal sinus involving cortical veins of H. Nakase et al. (11), hence producing a thrombosis model similar to that seen clinically. In the present model, we injected thrombin into the SSS together with the cortical veins, which led to cerebral edema and often hemorrhagic infarction, producing effects similar to those seen in patients with severe CVST. This model may provide a new means of studying the pathogenesis, as well as treatment therapies, for CVST. METHODS Animal Preparation Male Sprague-Dawley rats (280e310 g; Beijing Vitalriver Experimental Animal Co., Beijing, China) were studied. All procedures were conducted according to institutional guidelines and were in compliance with regulations formulated by the Animal Care and Use Committee, Capital Medical University of Beijing, China. A total of 49 rats were used in the experiment, and they were randomly divided into two groups: a study group (n ¼ 32) in which thrombi were induced in the SSS and cortical veins, and a sham-operated group (n ¼ 17). Five rats died from intracranial hemorrhage in the study group during/following the procedure, giving a study group mortality rate of 15.6%. A total of 44 rats survived; of the surviving rats, both groups were further divided into three groups and were used in statistical analysis. Rats allocated to subgroups were used for 1) edema studies (n ¼ 11 for study group; n ¼ 11 for sham group); 2) 2,3,5-triphenyltetrazolium chloride (TTC) staining (n ¼ 11 for study group; n ¼ 3 for sham group); 3) histologic examination (n ¼ 5 for study group; n ¼ 3 for sham group); and the data were analyzed 24 hours after induction of the thrombus. The animals were maintained on an alternating 12-hour light/dark cycle with free access to food and water. Surgical Preparation Anesthesia was induced by 5% isoflurane, and, throughout the surgery was maintained with 2%e3% isoflurane delivered in a mixture of 70% nitrous oxide and 30%
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oxygen by a face mask. During anesthesia, body temperature was monitored with a rectal probe and maintained at 37 C 0.5 C using a thermostatically controlled heating pad (Harvard Apparatus 50-7061-f, Holliston, MA, USA). In addition, PE-50 tubing was inserted into the left femoral artery for monitoring mean arterial blood pressures and obtaining blood samples to determined blood gases and glucose during the operation. Blood samples were obtained immediately before and at 30 minutes after the craniotomy, and at the beginning of SSS occlusion. The surgical procedure was performed as follows: a midline neck incision was made, the common carotid arteries were exposed bilaterally, and a suture was placed under the artery on each side. Next, the animal was placed in the sphinx position with the head fixed at midline using a stereotaxic frame (David Kopf Instruments, Tujunga, California, USA). A 2.0-cm midline skin incision was performed, exposing the calvaria. A longitudinal cranial window (8 4 mm), centered on the coronal and lambda sutures, exposing the sinus and bilateral parasagittal cortex, was created using a dental high-speed drill (Strong207B, Saeshin, Busan, South Korea) under microscopic control (Carl Zeiss, Inc., Berlin, Germany). The drill-tip was cooled continuously with physiologic saline to avoid thermal injury to the underlying cortex. Care was taken to avoid lesions of the SSS and bridging veins, and, after the window formation procedure, the dura mater was completely intact. The SSS was exposed and permanently ligated rostrally and caudally with 8-0 Prolene suture (Ethicon Endo-Surgery, Inc., Cincinnati, OH, USA). A microcatheter-shaped PE-50 (diameter, 0.35e0.38 mm) was inserted into the SSS rostral to the caudal ligation and visualized using an operating microscope (Nikon 80i, Nikon Instruments Inc., Melville, NY, USA). Then the microcatheter was sutured to the SSS to avoid leakage of the thrombin during injection. Both common carotid arteries were then temporarily occluded with suture; next, thrombin (100 mL, 50 IU/mL; ChangChun Grand Sinlo Pharmaceutical Co., Ltd., China) was injected into the SSS during 1 minute, by hand. The extended craniotomy, along the length of the SSS, allowed for visualization of both the thrombin injection and the induced thrombus
formation, providing assurance of maximal thrombus extension into the cortical veins. After the thrombin injection, the sutures were removed from the carotids to allow for reperfusion. The total carotid occlusion time was 1 minute. The microcatheter was withdrawn after a dark, solid blood clot was visible in the SSS, occurring approximately 5 minutes after thrombin injection. Upon completion of the procedure, the field was flushed with physiologic saline and the incision was closed. Animals were then returned to their respective cages and allowed to recuperate from anesthesia. Animals in the sham-operated group underwent anesthesia, had their femoral arteries cannulated, underwent surgery to form the longitudinal cranial window, and then the incision was closed. Neurological Evaluation Neurological evaluations were done in a blinded manner. The rats were subjected to a neurological evaluation before the surgery, as well as 0.5 and 24 hours after the surgery. Animals were scored as follows: 0 ¼ no observable neurological deficit (normal); 1 ¼ failure to extend left forepaw on lifting the whole body by tail (mild); 2 ¼ circling to the contralateral side (moderate); 3 ¼ leaning to the contralateral side at rest or no spontaneous motor activity (severe) (19). Magnetic Resonance Imaging Protocol The brains were observed for evidence of infarction and thrombus by magnetic resonance imaging (MRI) 24 hours after surgery. Animals were reanesthetized by an intraperitoneal injection of chloral hydrate (36 mg/100 g body mass) and placed in the prone position, with their heads inside birdcage radiofrequency coins (Siemens, Deerfield, IL, USA). The MRI imaging was performed using a 3.0-T Magnetom Verio syngo (Siemens). T1-weighted images, T2weighted images, diffusion-weighted images (DWI), and magnetic resonance venography (MRV) were acquired. (Tlweighted ¼ repetition time [TR] 500 ms, echo time [TE] 13 ms, field-of-view [FOV] 78 mm, 2.0-mm slice thickness, 11 slices; T2-weighted ¼ TR 4000 ms, TE 92 ms, FOV 64 mm, 2.0-mm slice thickness, 11 slices; DWI ¼ TR 3500 ms, TE 80 ms, FOV 94 mm, 2.0-mm slice thickness, 11 slices; MRV ¼ TR 46 ms, TE 5.14 ms, FOV 120 mm, 2.0-mm slice thickness, 30 slices.)
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Brain Water Content Twenty-four hours after CVST, the rats were sacrificed with chloral hydrate (40 mg/100 g body mass) administration, and the brains were subsequently removed. The cerebellum, pons, and olfactory bulbs were then removed from each brain and each cerebrum was weighed to determine its wet weight (WW). Subsequently, the brains were placed into a 110 C thermal oven for 24 hours, and then weighed to determine their dry weight. The formula (WW - Dry weight)/ WW 100% was used to calculate the water content percentage of each brain (6). 2,3,5-Triphenyltetrazolium Chloride Staining Rats were sacrificed with chloral hydrate (40 mg/100 g body mass) administration 24 hours after the surgery. Brains were harvested and cut into 2-mm thick coronal slices for TTC (Sigma-Aldrich, St. Louis, MO, USA) treatment. Brain slices were incubated in 1% TTC solution at 37 C for 20 minutes. Stained slices were fixed in 10% formalin solution. The decolorized area in each brain slice was determined using an image analysis program (Image J 1.44p; National Institutes of Health, Bethesda, Maryland, USA). The brain lesion volume was calculated by the product of average slice thickness (2 mm) and the sum of infarction area in all the six slices. The results were expressed as mean standard deviation. Histologic Examination Rats were sacrificed with chloral hydrate (40 mg/100 g body mass) administration, and then perfused cardiovascularly with physiologic saline and 4% paraformaldehyde in saline. The SSS and brains were collected. After paraffin embedding, 5-mm-thick coronal sections were cut and stained with hematoxylin-eosin (Sigma). The brains were viewed using a histologic light microscope (Nikon 80i), and observations were made regarding changes in the parenchymal tissue and SSS. Three sham-operated rats were used as controls for normal brain tissue and endothelial cells. Statistical Analysis Data were expressed as means standard deviation, and statistical analysis was performed with one-way analysis of variance and the t-test using SPSS for Windows,
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Table 1. Physiologic Parameters
Time
Mean Arterial Blood Pressure (mm Hg)
SaO2
pO2 (mm Hg)
pCO2 (mm Hg)
pH
Glucose (mg/dL)
Before sinus occlusion
91.7 4.9
98.6 1.03 116.66 7.66 46.78 3.00 7.42 0.06 132.3 10.6
Five minutes after occlusion
92.5 6.0
98.0 0.63 115.17 8.93 48.25 3.71 7.33 0.04 132.0 11.2
Values are expressed as mean SD. No significance was detected by analysis of variance analysis. pCO2, carbon dioxide partial pressure; pO2, oxygen partial pressure; SaO2, arterial oxygen percent saturation.
version 11.5 (SPSS Inc., Chicago, IL, USA). Statistical significance was set to P < 0.05. RESULTS Physiologic Variables In the study group, no significant differences were observed in the physiologic parameters (rectal temperature, blood alveolar oxygen tension, alveolar carbon dioxide tension, and pH, mean arterial blood pressure, glucose) as measured before and after venous occlusion (P < 0.05). Likewise, there were no significant differences in these variables when comparing the study group to the sham-operated control group (P < 0.05). Details regarding physiologic data are given in Table 1. Neurological Evaluation All animals exhibited a normal postural reflex (score ¼ 0) before surgery. Neurological scores in the study group were 1.74 0.59 at 0.5 hours after surgery, and 2.11 0.58 at 24 hours after surgery. There was no significant difference in neurological deficit at 0.5 and 24 hours after surgery. The study group displayed significant (P < 0.001) neurological deficits when compared with the sham-operated control group. The control group did not exhibit obvious neurological deficits (Figure 1). MRI Evaluation MRI confirmed that SSS occlusion and brain infarction were present in all animals, with hemorrhage occurring in 59% (16/27) of the animals. Figure 2 shows typical examples of the MRI findings. The MRV confirmed occlusion of the SSS after the operation (image E). The DWI image indicates the local brain infarction (image F).
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T1 and T2-weighted images indicate the presence of hemorrhagic infarction and brain edema, which contributed to a midline shift (images G and H). No brain lesions were observed in the images of sham-operated control rats (images AeD). Brain Water Content The brain water content of animals in the study group was 80.8% 0.55%, and in the sham-operated group was 78.8% 0.14%. There was a significant (P < 0.05) difference between the two groups (Figure 3). TTC Staining An SSS thrombus was detected in each of the study group animals, and infarctions were confirmed by TTC staining. Staining of the brain slices revealed a bilateral infarction in three animals and a unilateral infarction in eight animals. The brain lesion volume was 53.02 7.91 mm3 at 24 hours after the operation (Figure 4). No brain lesions were observed in the control group (not shown).
Figure 1. Time course of neurological deficit scores. In the study group, there was no significant difference between the neurological deficit scores at 0.5 and 24 hours after surgery. In comparison to the sham-operated control group, the study group displayed significant (*P < 0.001) neurological deficits. Sham-operated group, n ¼ 17; study group, n ¼ 27.
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Figure 3. The brain water content percentage in the sham-operated and study groups. There was a significant (*P < 0.05) difference observed between the two groups. Superior sagittal sinus thrombosus (SSST) indicates the study group animals with superior sagittal sinus thrombosis. Sham-operated group, n ¼ 11; study group, n ¼ 11.
The SSS was filled with blood, thrombus, and inflammatory cells determined by hematoxylin-eosin staining (Figure 5). DISCUSSION Several previous models of SSS have been developed (7, 10, 11, 15, 18, 19, 22, 23); however, in these models, the induced thrombus was confined to the SSS, with no thrombus formation in the cortical veins. Previous studies have shown that a thrombus, when restricted to the SSS, does not significantly alter cerebral blood flow, nor does it cause venous infarction and hemorrhage (10, 14). The present study demonstrates that an injection of thrombin
Figure 2. Magnetic resonance imaging (MRI) took place at the 24-hour time point. A magnetic resonance venography (MRV) image showing the superior sagittal sinus (SSS) in the normal shamoperated control rats (A, arrow). The arrow in E indicates filling defects at 24 hours after the operation. The MRV reveals the absence of a signal in the SSS and a normal flow signal in the anterior part of the SSS. Diffusion-weighted MRI (DWI) displays the hyperintense lesion (F, arrow). T1weighted MRI and T2-weighted MRI (G, H) show the infarction. In addition, the T2-MRI shows brain edema at 24 hours after the operation, especially in the left parieto-occipital lobes. Images from sham-operated control rats (AeD) show that no brain lesions were present. Superior sagittal sinus thrombosus (SSST) indicates the study group animals with superior sagittal sinus thrombosis.
Histologic Findings Hemorrhagic infarction, when present, and brain edema were both observed in the
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parenchyma of the parietal lobe. The image in Figure 5 illustrates that the distance between each neuron increased as a result of edema.
Figure 4. Triphenyltetrazolium chloridestained brain slices from animals in the study group. The unstained areas represent the infarction. Animal A exhibited an infarction on one side of the midline, and Animal B exhibited a bilateral infarction.
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Figure 5. Histologic images showing normal superior sagittal sinus (SSS) and brain tissue, and the effects of the induced thrombus. (A) SSS of a typical sham-operated animal. The sinus was surrounded by endothelial cells and no blood or thrombus was observed within the sinus. (B) SSS of a typical study animal. The sinus was filled with blood, thrombus (arrowhead) and inflammatory cells (arrow). (C) A typical coronal section of
into the SSS and cortical veins of rats can reliably model human CVST injury patterns, including infarction, parenchyma hemorrhage, and cerebral edema. In our proposed method, venous thrombosis was achieved by a combination of sinus ligation and thrombin injection. Ligation of the SSS produces venous stasis, a hemodynamic condition that favors thrombin activity and venous thrombosis (5). Furthermore, our proposed method involves bilateral occlusion of the common carotid arteries. This approach can reduce blood flow in the cortex by more than 60% (1), resulting in dramatically reduced cerebral perfusion pressure, thus allowing the thrombin to enter the cortical veins more easily. In addition, the thrombus in this animal model extended progressively into the cortical veins, a pattern often seen clinically in patients with CVST. Venous sinus thrombosis leads to disordered blood flow (reflux)
the SSS from an animal in the study group; the thrombus was in the SSS (arrow) and cortical vein (arrowhead). (D) Normal brain tissue from an animal in the sham-operated group. (E, F) Animals in the study group. Typical brain parenchyma bleeding (arrowhead) and edema (arrow). The distance between each neuron was wider than normal due to the edema. Scale bar (A, B, C), 1.25 mm; scale bar (D, E, F), 0.25 mm.
and increased venous pressure. As a result, the intracranial pressure increases and perfusion pressure decreases, leading to reduced cerebral blood flow. Spontaneous intracranial hypotension and low cerebral spinal fluid pressure have been shown to further accentuate thrombin activity and have been associated with cerebral venous thrombosis (8, 9, 12, 13, 24). An advantage of our proposed method is the ease at which the investigator can assess thrombus formation. It can be directly visualized, using microscopy, as a darkening in color of the dural walls comprising the SSS. Furthermore, no active bleeding was observed after the PE50 was removed. A potential shortfall of the model is that injection of thrombin into the SSS likely produces only a local hypercoagulable state. In our model, the hypercoagulable state was transient and local, whereas systemic hemodynamics
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likely remained relatively unchanged. As such, when using our rodent model, the investigator needs to be aware of the local hypercoagulability produced and how it differs from the systemic hypercoagulability often seen clinically. Headache is the most common symptom of CVST and occurs in approximately 90% of patients (4). Headache in patients with CVST is often reflective of increased intradural sinus pressure, a condition associated with parenchymal and cerebral edema (21). Venous thrombosis is associated with venous infarction and increased venous leakage, leading to vasogenic edema (2). Furthermore, circulatory stasis can impair the sodium pump and other blood-brain barrier functions, leading to cytotoxic edema (16). Total venous occlusion can lead to complications such as venous rupture and hemorrhage (16). Our method, unlike previously described models of venous
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thrombosis (7, 10, 11, 15, 22, 23), can induce many of the major complications seen in CVST, including infarction, hemorrhage, and edema. As such, we propose that our method is a more reliable model for assessing CVST and its complications compared with previously suggested models. REFERENCES 1. Choy M, Ganesan V, Thomas DL, Thornton JS, Proctor E, King MD, van der Weerd L, Gadian DG, Lythgoe MF: The chronic vascular and haemodynamic response after permanent bilateral common carotid occlusion in newborn and adult rats. J Cereb Blood Flow Metab 26:1066-1075, 2006. 2. Ducreux D, Oppenheim C, Vandamme X, Dormont D, Samson Y, Rancurel G, Cosnard G, Marsault C: Diffusion-weighted imaging patterns of brain damage associated with cerebral venous thrombosis. AJNR Am J Neuroradiol 22:261-268, 2001. 3. Einhaupl K, Stam J, Bousser MG, de Bruijn SF, Ferro JM, Martinelli I, Masuhr F: EFNS guideline on the treatment of cerebral venous and sinus thrombosis in adult patients. Eur J Neurol 17: 1229-1235, 2010. 4. Ferro JM, Canhao P, Stam J, Bousser MG, Barinagarrementeria F: Prognosis of cerebral vein and dural sinus thrombosis: results of the International Study on Cerebral Vein and Dural Sinus Thrombosis (ISCVT). Stroke 35:664-670, 2004. 5. Hassouna HI: Blood stasis, thrombosis and fibrinolysis. Hematol Oncol Clin North Am 14: xvii-xxii, 2000. 6. Kawai N, Kawanishi M, Okauchi M, Nagao S: Effects of hypothermia on thrombin-induced brain edema formation. Brain Res 895:50-58, 2001. 7. Kimura R, Nakase H, Tamaki R, Sakaki T: Vascular endothelial growth factor antagonist reduces brain edema formation and venous infarction. Stroke 36:1259-1263, 2005.
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8. Lai PH, Li JY, Lo YK, Wu MT, Liang HL, Chen CK: A case of spontaneous intracranial hypotension complicated by isolated cortical vein thrombosis and cerebral venous infarction. Cephalalgia 27: 87-90, 2007. 9. Lan MY, Chang YY, Liu JS: Delayed cerebral venous thrombosis in a patient with spontaneous intracranial hypotension. Cephalalgia 27: 1176-1178, 2007. 10. Nakase H, Heimann A, Kempski O: Local cerebral blood flow in a rat cortical vein occlusion model. J Cereb Blood Flow Metab 16:720-728, 1996. 11. Nakase H, Takeshima T, Sakaki T, Heimann A, Kempski O: Superior sagittal sinus thrombosis: a clinical and experimental study. Skull Base Surg 8:169-174, 1998. 12. Nardone R, Caleri F, Golaszewski S, Ladurner G, Tezzon F, Bailey A, Trinka E, Zuccoli G: Subdural hematoma in a patient with spontaneous intracranial hypotension and cerebral venous thrombosis. Neurol Sci 31:669-672, 2010. 13. Richard S, Kremer S, Lacour JC, Vespignani H, Boyer P, Ducrocq X: Cerebral venous thrombosis caused by spontaneous intracranial hypotension: two cases. Eur J Neurol 14:1296-1298, 2007. 14. Rottger C, Bachmann G, Gerriets T, Kaps M, Kuchelmeister K, Schachenmayr W, Walberer M, Wessels T, Stolz E: A new model of reversible sinus sagittalis superior thrombosis in the rat: magnetic resonance imaging changes. Neurosurgery 57:573-580, 2005. 15. Sarwar M, Virapongse C, Carbo P: Experimental production of superior sagittal sinus thrombosis in the dog. AJNR Am J Neuroradiol 6:19-22, 1985. 16. Schaller B, Graf R: Cerebral venous infarction: the pathophysiological concept. Cerebrovasc Dis 18: 179-188, 2004.
developing an experimental model. J Neurosci Methods 161:220-222, 2007. 19. Srivastava AK, Kalita J, Dohare P, Ray M, Misra UK: Studies of free radical generation by neurons in a rat model of cerebral venous sinus thrombosis. Neurosci Lett 450:127-131, 2009. 20. Stam J: Thrombosis of the cerebral veins and sinuses. N Engl J Med 352:1791-1798, 2005. 21. Tsai FY, Wang AM, Matovich VB, Lavin M, Berberian B, Simonson TM, Yuh WT: MR staging of acute dural sinus thrombosis: correlation with venous pressure measurements and implications for treatment and prognosis. AJNR Am J Neuroradiol 16:1021-1029, 1995. 22. Wang J, Ji X, Ling F, Luo Y, He X, Guo M, Li S, Miao Z, Zhu F, Xuan Y: A new model of reversible superior sagittal sinus thrombosis in rats. Brain Res 1181:118-124, 2007. 23. Wang J, Tan HQ, Li MH, Sun XJ, Fu CM, Zhu YQ, Zhou B, Xu HW, Wang W, Xue B: Development of a new model of transvenous thrombosis in the pig superior sagittal sinus using thrombin injection and balloon occlusion. J Neuroradiol 37:109-115, 2010. 24. Wang YF, Fuh JL, Lirng JF, Chang FC, Wang SJ: Spontaneous intracranial hypotension with isolated cortical vein thrombosis and subarachnoid haemorrhage. Cephalalgia 27:1413-1417, 2007.
Conflict of interest statement: This study was supported by New Century Excellent Talents from Ministry of Education (no. NCET-08-0625), Health Bureau of High Level Talent project (no. 2009-3-61), Chinese National Science Foundation (no. 30870854), and Board of Education Science and Technology Innovation Platform of Beijing (PXM2011014226-07-000070). Guangwen Li and Xianwei Zeng contributed equally to this work as co-first authors. Received 16 May 2012; accepted 19 November 2012
17. Sebire G, Tabarki B, Saunders DE, Leroy I, Liesner R, Saint-Martin C, Husson B, Williams AN, Wade A, Kirkham FJ: Cerebral venous sinus thrombosis in children: risk factors, presentation, diagnosis and outcome. Brain 128:477-489, 2005. 18. Srivastava AK, Gupta RK, Haris M, Ray M, Kalita J, Misra UK: Cerebral venous sinus thrombosis:
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