Abstracts

Rationale: The blood-brain barrier (BBB) contributes to brain homeostasis necessary for the correct function of neurons. While it was demonstrated that disturbance of this delicate equilibrium can be a cause of abnormal seizure activity, the events occurring at the time of seizures have not been thoroughly studied. We have evaluated the patterns of serum protein brain penetration and white blood cell (WBC) extravasation occurring at the time of acutely-induced seizures to help further understand the nature of these events. Methods: Hemispheric opening of the BBB was performed in rats by injecting 3 ml of mannitol (1.4M) in the internal carotid artery (ICA). Seizures occurrence was monitored by video-EEG. BBB damage was assessed by micro-angiography using a combination of FITC-Albumin/Evans Blue and quantified by fluorescent microscopy indexing the intensity of pixels x extravasation site in the frontal cortex, thalamus, and temporal cortex. For the evaluation of the pattern of WBC blood-to-brain distribution we used GFP transfected autologous cells. GFP-WBCs (3 to 4 x 106 cells) were injected together with Evans Blue in the ICA after BBBD. Quantification of WBC was performed by counting cells in the intravascular, perivascular, or parenchymal compartments in leaky and non-leaky vessels of corresponding areas of the brain. Results: EEG recordings confirmed the onset of abnormal activity in 20-25% of the rats immediately after osmotic opening of the BBB. A significant increase of the 8-15 Hz frequencies was recorded. Montage images of rat brain slides indicate a significant difference between protein extravasation in the two hemispheres (BBBD vs. non-BBBD). Generally, Evans Blue extravasated in the brain parenchyma to a greater extent compared to FITC-Albumin, being this difference more significant in the BBBD brain hemisphere. Spatial/intensity quantification of fluorescent signals confirmed the wide parenchymal spread of Evans Blue in all the areas analyzed. Injection of GFP-WBC + Evans Blue revealed the pattern of BBB damage and WBC distribution. The majority of WBC was confined within the vascular/perivascular space, with increased number of perivascular WBC in association with leaky vessels (intraluminally: 3.5 ± 0.3 and 2.5 ± 0.5 cells/vessel; perivascular 1 ± 0.5 and 5 ± 0.5 cells/vessel). Little or no parenchyma WBC was found among both leaky and non-leaky vessels in all brain areas analyzed. Conclusions: Our results depict the pattern of serum protein and WBC blood-to-brain distribution at time of acute seizures. We demonstrated the passage of serum protein through the BBB and the lack of WBC parenchymal invasion. These results suggest that the presence of WBCs in the brain parenchyma is not required to generate acute seizures.