Abstracts

Rationale: Serum auto-antibodies  directed against surface neuronal antigens are found in patients affected by encephalitis and seizures. How do antibodies (IgGs) access the brain parenchyma is still under debate. In this preliminary study, we aim to define the route of healthy IgGs accumulation in the brain at the cellular and sub-cellular levels and to study their acute capability to eventually modify the blood-brain barrier (BBB) permeability, trigger inflammatory processes or alter brain excitability. The possibility that cerebrovascular damage facilitates peripheral  IgGs  targeting neuronal epitopes remains also unexplored and represents the next step of this study. Methods: The capacity of healthy hIgGs (5mg/g) to alter basal neuronal activity and trigger BBB impairment and brain  inflammation was investigate in the in vitro isolated guinea pig brain, which represents a unique preparation in which the BBB is preserved up to 5 hours, as well as vascular and neuronal compartments (de Curtis et al.,  1998, 3:221–228; Librizzi et al., 2001, 14:174-178; Librizzi et al., 2006, 137:1211-1219). Control hIgGs were perfused intra-arterially in the isolated brain preparation for 30 min. Electrophysiological recordings were carry out continuously starting from 15 min before the control hIgGs perfusion and up to 60 min after the end of the treatment.  Then, brains were splitted into two hemispheres. One half was fixed to evaluate by IHC assays i) brain inflammation (e.g., gliosis, IBA1) and ii) brain cellular localization or uptake of hIgGs using tagged fluorescence. The other hemisphere was snap-frozen for WB analysis of sub-cellular localization of hIgGs.  Results: Preliminary results indicated hIgGs signal into the luminal space of cortical vessels at gray matter level and their extravasation into brain parenchima at white matter level.  Interestingly, ionized calcium binding adaptor molecule 1 (Iba1) staining appeared up-regulated and microglial cells moved from their ramified to fully active phagocytic form exclusively at white matter level. HIgGs signal co-localized exclusively with reactive microglial cells (Fig. 1; n= 10). Western blotting analysis performed on hippocampus and cortex homogenates obtained from control hIgGs treated vs untreated isolated brains, confirmed immunohistochemical data (Fig. 2; n=10). Electrophysiological recordings performed continuously during the experiment showed no changes of LOT-evoked potential in piriform cortex and CA1/DG regions (n=10). These results suggest a major propensity of white matter vessels to be impaired by continuous interaction with IgGs and demonstrate the pathogenicity of control IgGs following their access to brain parenchima. Conclusions: Our unique approach allows to track the pathophysiological neuro-vascular effects of hIgGs applied intra-arterially and to decipher their pathophysiological impact on brain inflammation and neuronal excitability. The next step forecasts to investigate whether acuter exposure to auto-Abs alters basal neuronal activity and triggers more abrupt inflammatory processes. Funding: Italian Health Ministry (Ricerca Corrente 2016)