The Permeability Change Of Blood-brain Barrier After Whole-brain Irradiation And The Expression Of Claudin-5 In C6 Glioma Rats

【Objective】To investigate the changes of BBB permeability in different regions of middle-late phase C6 glioma and their early dynamic changes after single whole-brain irradiation with different doses in rats, and to evaluate the mechanisms of the increased permeability of BBB/BBTB from ultrastructure of microvascular and the expression of tight junction protein Claudin-5. To explore the relationship between radiation and the blood-brain barrier opening,and establish a basis for glioma radiotherapy from animal experimental and the timely intervention of the possible radiation dose-domain and time-domain for chemotherapy.【Materials and Methods】(1) 10μl solution containing 1x106 glioma cells was stereotactically implanted into the right caudate nucleus to establish an experimental model of C6 glioma in 20 SD rats. Observe the survival status of rats, record the survival time, and perform the enhanced MRI scan at the 10th,14th,20th day after inoculation, respectively. Evaluation of the rate of glioma tumor modelling,the changes of tumor size,and the tumor growth duration to provide a stable source of glioma model for subsequent study.(2) 10 rats were equally divided into 2 groups, control group and tumor-bearing group. The rats with brain-bearing C6 glioma were sacrificed at the 20th day after inoculation, and then the samples were chosen from tumor tissues, BAT(tissues within 2 mm to the border of the tumor), BDT(cortical tissues more than 2 mm away from tumor border) respectively and the normal right caudate nucleus tissues of the control group. Using transmission electron microscopy(TEM) of exogenous lanthanum nitrate(La3+)-tracing to observe the BBB / BBTB ultrastructural features and the exudated extent of lanthanum nitrate,and determine the extent of BBB damage. (3) 10 rats were equally divided into 2 groups, control group and tumor-bearing group(20th day after inoculation). Using immunohistochemical method to detect the expression of claudin-5 in the normal brain and different expression of the above parts in tumor-bearing rats.(4) 120 tumor-bearing rats (20th day after inoculation) were randomly divided into 5 groups, namely sham irradiation group, 5Gy, 10Gy, 15Gy and 20Gy group. Groups were whole-brain irradiated with the corresponding single dose. Sham irradiation group (from the first and second part of the experiment), 5Gy, 10Gy and 20Gy groups were 20 rats, in which 8rats were sacrificed at d1 after irradiation,4 rats were using transmission electron microscopy of exogenous lanthanum nitrate(La3+)-tracing to observe the BBB/BBTB permeability of BAT department and using immunohistochemical method to detect the expression of claudin-5, repectively.15Gy group was 60 rats, in which 8 rats were sacrificed at d1, d3, d7, d14, d21, d28 after irradiation, which were perfored to the above mentioned detection; each of the 12 rats remained to natural death. Observed and recorded the survival of irradiated rats, and survival time.【Results】(1)Using stereotactic methods to establish the C6 glioma model was exact and reliable, with modelling rate of more than 97%, and without distant and extracranial metastasis. The early symptoms of tumor-bearing rats, included declined movement, declined offense, depressed, and so on. The advanced symptoms included paralysis, orbital hemorrhage, epilepsy and cachexia, and reduced body weight. MRI examination showed the tumors were round or nearly round, arranged with long T1 and T2 signal, most of which were ring-like enhancement.(2) Using TEM of exogenous La3+-tracing to observe the C6 glioma tumor- bearing rats. In samples of tumor department, La3+ could be seen acrossing the TJ of vascular endothelial cells and basement membrance (BM)to penetrate extra-vascular and brain tissue space in a wide range. Part of capillary endothelial cells and nerve cells lose their normal form and damage their structure, with a large number of La3+ deposited in the nucleus inside and outside.In BAT,La3+ exuded to the local BM, most of which lie in the endovascular and no La3+ seeped to brain tissue gap.In BDT and the control group NBT ,La3+ distributed among endovascular and the structure of vascular endothelial cell TJ was integrity. The BM was lineally continuous, and no La3+ was deposited in cerebral vascular endothelial cytoplasm and extra-vascular.④Statistical analysis showed: tumor department> BAT> BDT, NBT (P<0.05), and no significant difference was found between the BDT and NBT groups.(3) Using TEM of exogenous La3+-tracing to observe the BBB permeability in BAT of the tumor-bearing rats after irradiation with different doses. In BAT of 5Gy group, La3+ exuded to the local BM, most of which lie in endovascular, and cell morphology hadn't obvious abnormalities.In BAT of 10Gy group,La3+ exuded to a wide range of BM, exudation was increased and tight junctions were opened. The high electron density of small particles of lanthanum seeped to the brain tissue space, endothelial cell swelled, and some remained in the endovascular.In BAT of 15Gy, 20Gy group,La3+ seeped to the BM, extra-vascular, the space inside the brain tissue and cells, with endothelial cell swelling and brain edema. Statistical analysis showed that 20Gy, 15Gy> 10Gy> 5Gy, 0Gy (P <0.05), and there is no significant difference between the group of 0Gy and group 5Gy, no significant difference was also found between and the group of 15Gy and 20Gy.(4) Using TEM of exogenous La3+-tracing to observe the BBB permeability changes in BAT of 15Gy group rats at different time points after exposure. At 1,3 days after exposure, La3+ exuded to the BM , extra-vascular, the space inside the brain tissue and cells, a little remaining in the endovascular.At 7 days after exposure, La3+ exuded to the BM and brain tissue space less than the previous days, not exuding into cells.At 14 days after exposure, La3+ exuded to the local EM,but not in extra-vascular, the brain tissue space and cells inside, the majority remaining in the endovascular.At21,28days after exposure, La3+are located in endovascular only, not leaking to the BM,extravascular and the brain interstitium. Statistical analysis showed that 1d, 3d> 7d> 14d> 21d, 28d (P <0.05), no significant difference was found between the 1d and 3d groups, and the 21d and 28d groups were identical to the results.(5) Claudin-5 in both brain tissue of tumor-bearing and control rats showed yellow or brown along the vascular endothelial cells slurry, which was positive expression. The expression of claudin-5 in the brain tissue of control group was rich and continuous,showing medium-intensive expression.Claudin-5 of the tumor department was diminished expression, showing mild expression, and the expression along the blood vessels were interrupted. Claudin-5 expression in BAT was decreased than in the control group brain tissue,showing moderate expression, with vascular distortion and deformation, but still continuous state.Claudin-5 expression in BDT was similar to the normal brain tissue of control group. Statistical analysis showed that tumor department> BAT> BDT, NBT (P <0.05), and the BDT, NBT had no significant difference between two sets of results.(6)The expression of Claudin-5 in BAT of the tumor-bearing rats after irradiation with different doses was following up.Claudin-5 expression in BAT of 5Gy Group was decreased than in the control group brain tissue,showing moderate expression, with vascular distortion and deformation, but still continuous state. 10Gy group BAT Department could be seen moderate expression, combining with vascular distortion, deformation and continuous expression state. 15Gy, 20Gy group BAT Department could be seen mild expression, showing intermittent and debris-like expression along the vessels. Statistical analysis that 20Gy, 15Gy> 10Gy> 5Gy, 0Gy (P <0.05), and there is no significant difference between the group of 0Gy and group 5Gy, no significant difference was also found between and the group of 15Gy and 20Gy.(7)The expression of Claudin-5 in BAT of the tumor-bearing rats showed at different time points after irradiation with 15Gy was following up.Claudin-5 expression was mild at 1d and 3d after irradiation, showed intermittent expression along the vessels and debris-like expression.Claudin-5 expression was light and moderate at 7d after exposure, combined with vascular distortion, deformation and continuous expression. It was moderate expression 14d after exposure , showed vascular distortion and deformation, but still continuous state.It was medium-intensity expression at 21d and 28 days after irradiation with partial occlusion of small blood vessels, appeared to express both a row and debris. Statistical analysis: 1d, 3d>7d>14d>21d, 28d (P<0.05), and there is no significant difference between the group of 1d and group 3d, no significant difference was also found between and the group of 21d and 28d.(8)The lanthanum nitrate exudation correlates negatively with the expression of claudin-5 in C6 glioma different regions,including tumor department,BAT and NBT,and correlation coefficient was rs=-0.599 and P=0.04.The degree of La3+ exudation and claudin-5 expression in the BAT department of the tumor- bearing rats showed a significant negative correlation,when 1d after radiotherapy with different doses and at different time points after 15Gy irradiation,which correlation coefficient was rs=-0.678,P=0.001 and rs=-0.571, P=0.004,respectively。 (9)The MeST of sham irradiation group, 5Gy group, 10Gy group, 15Gy group and 20Gy group was 24d, 30.5d, 37.5d, 49d and 63d, and the MST of each group was 24.3±3.7d, 33.0±7.4d, 39.8±10.0d, 65.3±47.1d and 84.4±58.8d,respectively.IN each exposure groups the %ILS of MeST was 27.1%, 56.3%, 104.2% and 162.5%, and the %ILS of MST was 35.8%, 63.8%, 168.7% and 247.2%, respectively. The survival time of the four exposure groups were significantly different from the sham exposure group (P <0.05).【结论】(1)The C6 glioma model in rats were established successfully, which showed invasive growth property and similar to human glioma. The model implanted with 1x106 C6 gliomaous cells sterotactically into intracerebral was used for the treatment of experimental studies, and the appropriate time to the treatment was 2 to 3 weeks after cells inoculation.(2)The microvessel ultrastructural changes of C6 glioma is the structural basis for opening up the blood-brain barrier, and tight junctions opening up is the main reason to increase microvascular permeability.(3)C6 glioma endothelial ultrastructural changes in different regions are varying with the degrees of damage to barrier function. The more shorter distance from tumor department, the more lanthanum nitrate exudation, the lower expression of claudin-5, the more serious damage in cell TJs, and the more increased in microvascular permeability. The blood-brain barrier of BDT Department was similar with normal brain tissue with low permeability and stability,in which no lanthanum nitrate is exuded and claudin-5 expression is rich. Tumor surrounding exists a different levels of barriers ,which indicates barrier existing around the tumor.(4)The direct invasion of C6 glioma cells can reduce the expression of tight junction protein claudin-5 at endothelial cells. Claudin-5 lost from the cell is the molecular basis of TJ opening ,which is an important reason for TJ destruction leading to BBTB-permeability increased.(5)The BBB of exposed rats showed pathological changes, and the expression of claudin-5 was further reduced in rats after irradiation,which showed that radiation could induce the blood-brain barrier tight junction damaged increasely and reduce the role of BBB function.(6)The lanthanum nitrate exudation correlates negatively with the expression of claudin-5 in C6 glioma at different regions.The degree of La3+ exudation and claudin-5 expression in the BAT department of the tumor-bearing rats showed a significant negative correlation, when 1d after radiotherapy with different doses and at different time points after 15Gy irradiation.( 7 ) With dose and time changes during whole-brain radiotherapy, BBTB permeability of tumor-bearing rats changed regularly. BBB began opening up 1d after single irradiation 10Gy, and significantly after single irradiation 15Gy, 20Gy, in which the two doses have not differences. BBB is gradually resuming 2 weeks after irradiation 15Gy, showing dose-and time-dependent. With the role of radiation to treat cancer, radiation can extend survival time.