Blood-Brain Barrier Permeability Change And Vegf Expression Up-regulating In A Recurrent Headache Rat Model

Objectives1. To observe the changes of mechanical thresholds in a rat model of recurrent headache via episodic dural stimulation;2. To discuss the effect of recurrent headache on the expression of VEGF and BBB permeability change;3. To observe whether specific anti-migraine drug sumatriptan can counter the effect of recurrent headache.Methods1. Building of recurrent headache rat modelAccording to the protocol described by Oshinsky, rats were anesthetized and a1mm craniotomy was performed above the junction of the superior sagittal and transverse sinuses to exposure the dura. A plastic cap with a sterile stainless steel cannula was inserted.20μl of IS was pumped onto dura3times per week on Monday, Wednesday and Friday. Control group received administration of20μL of PBS. The rats in treatment group were treated with sumatriptan (300ug/kg, intraperitoneal) at the same time as IS was applied onto the dura every time.2. Behavioral testingElectronic von Frey was employed to test the withdrawal response of face to mechanical noxious stimuli. After the surgery, the animals were allowed to recover for one week. Before IS was pumped, baseline thresholds were determined. Then, the pain thresholds were tested before rats were administrated with IS for that day. Meanwhile, the thresholds were also done before and at0.25,0.5,1,1.5,2, and4hours after3,6and9IS stimulation.3. BBB permeability changes were measured by albumin immunofluorescence (IF) staining and Evans blue tracer. The expression of VEGF was measured by double labeling and Western blotting.Results1. IS induced mechanical hypersensitivityDuring the first week (IS pumping times1-3), there was a small decrease in mechanical pain threshold, but this was not significantly different when compared to control group (ANOVA, P>0.05). After the fourth pumping, the rats in IS group began transition from normal pressure thresholds to an hypersensitive state, and thresholds were significantly lower (ANOVA, P<0.05). Following8IS pumping, the thresholds decreased to below2g. The pain thresholds were lowest at30min after3,6and9IS applications. However, mechanical thresholds reduction induced by3and6applications gradually subsided after1h. Mechanical hypersensitivity persisted even at4h after9applications.2. IS induced BBB permeability changesThe results of albumin IF staining showed albumin could not be detected in control and3IS groups. In6IS group, there was mild IF staining. However, strong green fluorescent was observed in brain parenchyma around blood vessel, which demonstrated BBB permeability increase and albumin extravasation. Sumatriptan treatment reduced the BBB damage.EB was employed to evaluate the integrity of BBB among control,9IS and treatment groups, and the results were consistent with albumin IF staining. There was no EB extravasation in control group, but was abundantly present around the blood vessel after9applications. Sumatriptan reduced EB leakage in rats. Quantitative analysis revealed the EB contents of the brain tissue in9IS group was significantly higher than that in control group (0.96±0.25ng/mg VS3.45±0.42ng/mg, P<0.05), and it decreased to1.14±0.34ng/mg after sumatriptan treatment, it was significantly different when compared to9IS group (P<0.05)3. IS induced the changes of VEGF expressionIF analysis showed the expression of VEGF protein was few, which was been elevated after IS stimulation, especially after9IS stimulation, it was markedly increased. There were numerous red fluorescent positive cells. Double-labeling demonstrated positive cells were mainly neurons and VEGF were present in cytoplasm of neurons, which indicated IS induced numerous expression of VEGF in neurons. The numbers of positive cells were greatly reduced after sumatriptan administration.The results of Western blotting indicated the expression of VEGF protein mildly increased in3and6IS groups, but no significant differences when compared with control group (P>0.05). A significant up-regulation was detected after9applications (P<0.05). Sumatriptan reduced the expression of VEGF (P<0.05, compared to9IS group).Conclusions1. Repeated IS stimulation on dura produced a chronic state of trigeminal hypersensitivity;2. Repeated IS stimulation up-regulated the expression of VEGF protein participating in BBB permeability increase;3. Sumatriptan treatment inhibited transmission of noxious signaling and prevented the over-expression of VEGF as well as BBB leakage.