动物实验,硬脑膜动静脉瘘 z-bo 2020-09-02 410

　　Background: Dural arteriovenous fistula (DAVF) refers to the abnormal connection between arteries and meninges or dural sinuses or cortical veins. This is an intracranial vascular malformation. Dural arteriovenous fistulas account for about 10% to 15% of intracranial vascular malformations, 6% of supratentorial arteriovenous malformations and 35% of mandibular venous malformations. Although dural arteriovenous fistulas can occur anywhere, these vascular malformations are most commonly found in the cavernous sinus, lateral sinus, sigmoid sinus, and sagittal sinus (SSS). The main form of DAVF treatment is intravascular embolization. The cause of DAVF is unknown. There is a unique acquired reason. There is a view that dural arteriovenous fistula is a congenital disease caused by intracranial arteriovenous malformations and dural vascular abnormalities. In many clinical trials, the formation of dural arteriovenous fistula may be caused by brain trauma, sinusitis, sinus thrombosis, intracranial tumors, brain surgery, hypercoagulation, abnormal blood molecules, etc. display. DAVF is an acquired vascular disease. It is believed that acquired DAVF is due to the close relationship between DAVF and sinus thrombosis. Many researchers have established a total cervical external anastomosis (CCA-EJV) in a rat model of sinus hypertension. 1) High sinus pressure may induce DAVF; 2) DAVF disappears after automatic elimination of great sinus pressure; 3) Venous pressure in hypertensive group increased significantly 28 days after surgery; 4) Sinus thrombosis is a risk factor for high sinus pressure. An important reason for the formation of DAVF is the increase in intracranial sinus pressure. There are two theoretical explanations. One is the open "physiological arteriovenous anastomosis", and the other is "vascular endothelial growth factor-induced dural neovascularization". The purpose of this study is to use rabbit model pressure to induce high intracranial vein formation to study the mechanism of DAVF formation in more detail. In this study, we used cca-pfv anastomosis to generate a model of high intracranial venous pressure, and successfully formed DAVF. For the first time, the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor was found in a rabbit model.

　　Methods: Experimental animals: The study was approved by the welfare ethics committee. 100 Japanese rabbits (weight 2.0-2.5kg). Anesthesia: Inject 1% sodium pentobarbital (25 mg/kg) into the ear vein (EV) to induce anesthesia, such as arteriovenous anastomosis, carotid artery catheter placement, and sample collection. 1% sodium pentobarbital was injected into the carotid artery through a cerebral angiography catheter. Animal preparation: Grouping of experimental animals: 50 Japanese long-eared rabbits were randomly divided into 5 groups, A-E.A group (control group) sham operation (n = 10). In group B, an end-to-end anastomosis (EEA) of the right common carotid artery (CCA)-posterior facial vein (PFV) was performed (n = 10). In group C, the right common carotid artery (CCA)-posterior facial vein (PFV) and left external jugular vein (EJV) were performed (n = 10). In group D, the right common carotid artery-posterior cca-pfv end-to-side anastomosis was performed (n = 10). In group E, end-to-side anastomosis with cca-pfv of the posterior part of the right common neck and EJV ligation (n = 10) was performed. Two rabbits were randomly selected from each group on 7, 14 and 90 days after the operation, and the number of dural microvessels was counted after the ink injection. 90 days after the operation, four rabbits in each group were selected for digital subtraction cardiovascular angiography to observe the formation of DAVF. In addition, 50 Japanese white rabbits were randomly divided into 5 groups, namely the A-E group. Group A (control group) sham operation (n = 10). Group B-E (n = 40) underwent end-to-end anastomosis (EEA) of right common carotid artery (CCA)-posterior facial vein (PFV) and ligation of left external jugular vein (EJV). For groups B, C, D, and E (n = 10), specimens were collected for immunohistochemistry (6 in each group) and Western blot at 1, 2, 3, and 90 days after surgery. we. Analysis (4 per group). Model preparation: The animals were fasted for 10 hours before the operation and there was no drinking water restriction. Inject 1% sodium pentobarbital (25 mg/kg) into the left ear vein EV. After anesthesia, the rabbit was fixed on the operating table. Shave the neck, disinfect with iodine, and make a skin incision on the neck. Perform the following steps under the microscope.

　　1. Group A: Anatomically separate the bilateral external jugular veins. The proximal end is the anterior 1 cm ligature at the intersection of the anterior and posterior veins and the external jugular vein, and the distal end is the 1 cm ligation at the intersection of the anterior and posterior veins and the external jugular vein. This is a ligature. Expose the right carotid artery triangle to clearly separate the 2 cm CCA. Insert the 24# intravenous (IV) catheter into the posterior facial vein (PFV) and CCA respectively, and connect to the invasive pressure gauge to measure the experimental pressure of CCA and PFV. Apply a small amount of penicillin locally and suture the incision.

　　2. In group B, the bilateral external jugular veins and the right CCA were separated (the same length as the sham operation group). Measure normal arterial pressure and PFV pressure. Ligate the front end of the right external jugular vein (0.5 cm at the junction of the anterior and posterior anterior vein and the external jugular vein). Ligate the distal anterior vein (AFV). Clamp the PFV with a vascular clamp, and cut approximately 3 cm before the posterior facial vein (PFV) intersects with the external jugular vein. Trim the proximal end of the CCA, ligate and cut the distal end, and flush the vessel lumen with 1 mg/ml heparin/saline. The remaining CCA and PFA were stained with methylene blue and washed with 1 mg/ml heparin/saline. The end-to-end CCA and PFV anastomosis were performed with 9-0 sutures. After the anastomosis, confirm that the anastomosis is unobstructed. After the anastomosis, the PFV pressure was measured, a small amount of penicillin was applied to the incision, and the wound was sutured.

　　3. Group C separates right CCA, EJV and left EJV. The left EJV was ligated with 4-0 suture. Other operations are the same as group B.

　　4. In group D, separate the bilateral external jugular vein and the right CCA (the separation length is the same as that of the sham operation group). Measure normal arterial pressure and PFV pressure. Ligate the anterior part of the right EJV and the distal end of the AFV. Clamp the PFV and cut approximately 3 mm at the intersection of PFV and EJV. The vascular cavity was washed with 1 mg/ml heparin/saline. Use two container clamps to clamp the CCA, the distance between the two clamps is about 1.5 cm. Use micro scissors to cut along the wall of the container. The length of the cut is 1.5 times the pipe diameter. Wash the blood vessel lumen with 1 mg/ml heparin/saline. The CCA cut and PFV stump were stained with methylene blue, and washed with 1 mg/ml/heparin saline. An end-to-side anastomosis was performed between the common carotid artery and PFV with 9-0 sutures. Check the smoothness of the anastomosis. Measure the PFV pressure after anastomosis, and suture the incision with a small amount of penicillin.

　　5. Group E: Independent CCA, with EJV on the right and EJV on the left. The left EJV was ligated with 4-0 suture. Other operations are the same as group D. Pressure measurement: The right common carotid artery (CCA) of 50 rabbits in the 5 groups was dissected, and the CCA pressure was measured.

　　Common carotid artery blood pressure measurement: Peel off the CCA about 2 cm, insert a 24# IV catheter from the tail, fix it with a blood vessel clip, and then connect it to a non-invasive blood pressure measurement device. At the heart level, the pressure is adjusted to zero. After the reading is stable, take a picture and record it. Facial venous pressure measurement: The posterior vein (PFV) of 50 rabbits in 5 groups was stripped, and the PFV pressure was measured. The total length of the right external jugular vein, anterior vein and posterior vein is approximately 2 cm. The 24#IV catheter is inserted into the PFV through the venous valve, fixed and connected to the invasive pressure gauge. At the heart level, the pressure is adjusted to zero. After the reading is stable, take a picture and record it. Measurement of facial venous pressure after anastomosis: 40 rabbits in the B-E group confirmed the patency of the cca-pfv anastomosis before measuring the PFV pressure and anastomosis. The 24#IV catheter is inserted into the PFV through the venous valve, fixed and connected to the invasive pressure gauge. At the heart level, the pressure is adjusted to zero. After the reading is stable, take a picture and record it. Ink perfusion: On the 7, 14 and 90 days after CCA-PFV anastomosis in groups B-E and A, two rabbits in each group were selected for neck perfusion. Count the dural microvessel density. DSA test: 90 days after the operation, 4 rabbits in each of the 5 groups were selected for DSA test. Specific steps are as follows:

　　Place the catheter in the carotid artery. The animal was anesthetized by injecting 1% sodium pentobarbital (25 mg/kg) through ear vein, and the rabbit was placed on its back and fixed on the operating table. Dissect along the original neck incision. Carefully dissect the right arteriovenous anastomosis site. Many new contexts are being formed. After confirming that there is no obstruction at the anastomosis, measure the postoperative facial vein pressure. Correct the anastomosis site. Remove the CCA on the left and place a 24G IV catheter. The catheter was filled with 1 mg/ml heparin and the neck incision was closed.

　　Collect DSA images: Inject 1% sodium pentobarbital through CCA and fix the rabbit's back on the DSA table. Adjust the position of the machine and inject iohexol-300 through the CCA contrast media catheter (2ml/s, 3ml). X-rays before and after shooting. Immunohistochemistry: Control group, 7th, 14th, 21st, and 90th day groups, each of 6 rabbits HIF-1α and VEGF immunohistochemistry in occipital lobe and dural specimens. I collected the occipital cortex because of this The area is first affected by the anastomosis, resulting in increased intracranial pressure and can be more easily checked. The immunohistochemical sections of the tissue were fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin (HE). Tissue sections were incubated with the following main antibodies: vascular endothelial growth factor (VEGF) (C) (1:100), hypoxia-inducible factor-1α (1:200), overnight at 4°C. Next, they were incubated with horseradish peroxidase-labeled mouse anti-rabbit secondary antibody (1:1000). 3,3'-Diaminobenzidine (DAB) method is used for immunohistochemical analysis. All samples were observed under an inverted microscope and Olympus BX51 camera system. The percentage is the number of positive cells divided by the total number of cells. The standard used to express the level assignment value is: -= 0%, + =\→ 0%-25%, ++ = 26%-50%, +++ = 51%-75, %, ++++ = \→ 75%.

　　Western Blotting: Four rabbits from the control group were taken from the occipital cortex and dura mater, and Western blotting was performed on the 7, 14, 21, and 90 day groups of rabbits. The sample was dissolved in RIPA buffer, and the concentration of the protein extract was measured by the Bradford method. 40μg total protein was subjected to SDS-PAGE electrophoresis, transferred to a 10% PVDF membrane, and incubated in a TBS solution containing 0.1% Tween 20 and 5% skimmed milk powder. Next, treat with specific vascular endothelial growth factor VEGF (C-1) primary antibody (1:200), HIF-1α primary antibody (1:200) and α-tubulin (molecular weight 52 kDa, 1:3000) membrane. Incubate overnight at 4°C. The membrane was incubated with mouse anti-rabbit antibody labeled with horseradish peroxidase (1:1000). Use enhanced chemiluminescent horseradish peroxidase substrate to observe the immune response area. The results represent 3 independent experiments. The quantitative analysis was performed by the QuantityOne software of GE Image Scanner.

　　Result: The animals in group B and C survived after the operation. Due to high intracranial venous pressure, two rabbits in group D and E died within 3 days after the operation. The anastomotic patency rate of group B was 80%, group C was 90%, group D was 60%, and group E was 50%.

　　Perioperative blood pressure: There was no significant difference in the frequency of anastomoses in the B-E group after surgery. There was no significant difference in postoperative venous blood pressure between the sham operation group (group A) and baseline. Compared with the baseline after surgery and before slaughter, the venous pressure of the animals in the B-E group increased significantly. Compared with the control group after and before the operation, the venous blood pressure increased significantly after the operation. The venous blood pressure of animals in groups B and C was significantly higher than that in groups D and E. There were no statistically significant differences between group B and group C and between group D and group E.

　　Brain ink perfusion: Observe and compare the dural microvasculature from the convex sagittal sinus with a width of 2 mm and the lateral sinus with a width of 3 mm or more. The number of dural capillaries is microvessels per square millimeter. The number of dural microvessels in the control group was 12±2 mm 2. The number increased slightly to 15±2 mm 14 days after surgery. 2. After 90 days, the number of meningeal microvessels increased significantly, the number of groups B, C, D and E They are 36±4 square millimeters, 39±5 square millimeters, 33±3 square millimeters, and 35±4 mm respectively. 2. DSA test: 90 days after surgery, select 4 animals from each group in the AE group for DSA test. The DSA image of the arterial phase is shown in Figure 4, and the venous phase is shown in Figure 5. In group A, the blood vessels were unobstructed, there were no abnormal blood vessels, and the blood circulation time was about 11-15 seconds. In groups B, C, D and E, vascular tortuosity, dural arteriovenous fistula and AVF were observed in the ear and eye area. More than one arteriovenous fistula was observed in some animals. 22 cases of arteriovenous fistulas were found in 16 rabbits. The formation rate of DAVF was 43.75% (7/16) because 7 of them had DAVF. DAVF is mainly located in SSS, cavernous sinus and lateral sinus. DAVF is excreted through the SSS, lateral sinuses, and posterior facial veins. The arteriovenous fistula in the eye drains through the anterior vein. The ear arteriovenous fistula drains through the posterior vein, and the jugular fistula drains through the posterior vein. In groups B, C, D and E, the cycle time is extended (about 32-40 seconds). The circulation time in the venous phase was significantly extended, but the circulation time in the arterial phase was not extended (about 5 seconds).

　　Immunohistochemical method was used to detect the expression levels of VEGF and HIF-1α: VEGF expression in the occipital cortex and vascular endothelial cells in the 1 week and 2 week groups, the control group, and the 3 week and 90 day groups was much higher than that. Compared with the control group, the expression level of VEGF in SSS increased significantly in the 2-week-old animals. In the 1-week group, the expression of HIF-1α in the occipital lobe, mammary gland blood vessels and SSS was higher compared with the control group and the 2, 3, and 90-day groups. Western blot analysis of VEGF and HIF-1α expression levels: The expression of VEGF in the occipital dura mater is similar to the expression of HIF-1α in the occipital cortex and SSS area. After 1 week, 2 weeks, 3 weeks, 90 days and the control group, the expression peak appeared. On days 1, 2, 3, and 90, the average expression levels of VEGF expression in the occiput of the control group were 10.1, 27.2, 34.1, 16.9 and 11.8, respectively. The average expression levels of HIF-1α in the occipital lobe of the control group were 9.1, 35.6, 24.7, 20.5 and 10.1 under the skin for 1 week, 2 weeks, 3 weeks and 90 days, respectively. In the control group, the SSS area was 1 week, 2 weeks, 3 weeks and The average expression levels of HIF-1α at 90 days were 9.4, 35.6, 26.7, 17.7 and 10.6, and the comparison between the two groups was statistically significant.

　　Conclusion: The results of animal model experiments show that intracranial hypertension is a key factor in the formation of DAVF. Insufficient cerebral perfusion pressure caused by cerebral ischemia plays an important role in this process. Increased intracranial venous pressure leads to increased expression of HIF-1α and increased expression of VEGF.