Effects Of EMP Exposure On The Permeability Of Blood-Retinal Barrier And Its Mechanism

Electromagnetic pulse (EMP), a kind of pulse wave with an ultra-wide spectrum, has more severe biological effects on human beings contrasting to the continuous wave. Accordingly, people are getting concerned with effects of EMP exposure on human health. Eyes are sensitive to EMP exposure. Blood-retinal barrier (BRB) is critical for maintaining low permeability and retina homeostasis, which is fundamental for proper retinal function and vision. Tight junctions are important for sustaining the integrity of BRB. In this study, we evaluated the effect of EMP exposure on the functional integrity of BRB, examined the alterations of tight junction proteins of BRB and investigated whether mitogen-activated protein kinase (MAPK) pathway was involved in this procedure.In the in vivo study, Male Sprague-Dawley (SD) rats were sham exposed or exposed to EMP at200kV/m for200pulses. The alterations of BRB permeability were analyzed by observing fluorescence microscopy of retinal stretched preparations and quantitatively assessed by using evans blue (EB) as a tracer. Endogenous albumin was also assessed to analyze the BRB permeability by western blots. The results turned out that, red fluorescence of EB in the sham group was confined to the retinal vessels and the vascular structure was clear. After0.5h from the exposure, slight extravasations of EB could be seen out of some retinal vessels and structures of those vessels were still clear. After3to6hours, flaky red fluorescence of EB could be seen on some area of the retinal stretched preparation and the course of the vessels were not clear. After12to24hours, few red fluorescence of EB (not flaky) out of the vessel with clear vascular structure could be observed. EB quantitative assessment demonstrated that compared to the sham group, EB leakage increased at0.5h,3h,6h,12h,24h after the EMP exposure and reached to the maximum during3-6h after the exposure. At48h after the exposure, the level of EB leakage recovered to that of the sham group. The results of albumin western blots showed that the leakage of albumin increased at0.5h,3h,6h,12h after EMP exposure and reached to the maximum during3-6h. After the time-effect relationship of EMP exposure on BRB permeability was identified, the expressions of tight-junction-associated proteins and some signaling molecules of MAPK pathway were measured by western blots. The results showed that compared to that of the sham group, the expression of occludin decreased at3h and6h after EMP exposure; the expression of claudin-5decreased at0.5h,3h,6h,12h after the exposure; the expression of ZO-1remained unchanged. Contrasting to that of the sham group, the expression of p-p38, p-JNK, p-ERK increased at0.5h after the EMP exposure while the expression of p38, JNK, ERK did not alter.To simulate the in vivo situation of BRB, RF/6A and C6were co-cultured in transwell. After being established, the co-cultured in vitro model were sham exposed or exposed to the EMP(200kV/m,200pulses) to investigate the effect of EMP exposure on the model. The permeability of the model was analyzed by testing transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) permeability. The results showed that TEER value decreased at0.5h,3h,6h after the exposure and HRP permeability increased at0.5h,3h,6h after the exposure compared to that of the sham group. The consistent results demonstrated that after the exposure, the permeability of the in vitro model transiently increased. The expressions of tight-junction-associated proteins and MAPK pathway molecules of RF/6A were then measured by western blots. The results showed that compared to that of the sham group, the expression of occludin and claudin-5decreased at0.5h,3h,6h after the EMP exposure while ZO-1expression remained unchanged, p-p38, p-HSP27, p-JNK and p-ERK all increased at0.5h after the exposure. To further study the mechanisms of the procedure, RF/6A were pretreated with specific inhibitors of p38-MAPK, JNK and MEK1/2which respectively inhibited the phosphorylation of p38, JNK and ERK1/2before being exposed to EMP exposure. The specific inhibitor of p38-MAPK inhibited the increase of p-p38and p-HSP27. The decreases of tight junction proteins occludin and claudin-5induced by EMP exposure were also inhibited. So we concluded that p38-MAPK pathway was involved in the mechanism of the increase of BRB permeability induced by EMP exposure.To conclude, these results suggested that the alterations of occludin and claudin-5played an important role in the disruption of tight junctions, which might lead to the transient breakdown of BRB induced by EMP exposure. P38-MAPK pathway was involved in this procedure through phosphorylation of signaling molecules.