Ultrasounds induce blood-brain barrier opening across a sonolucent polyolefin plate in an in vitro isolated brain preparation

The blood-brain barrier (BBB) represents a major obstacle to the delivery of drugs to the central nervous system. The combined use of low-intensity pulsed ultrasound waves and intravascular microbubbles (MB) represents a promising solution to this issue, allowing reversible disruption of the barrier. In this study, we evaluate the feasibility of BBB opening through a biocompatible, polyolefin-based plate in an in vitro whole brain model. Twelve in vitro guinea pig brains were employed; brains were insonated using a planar transducer with or without interposing the polyolefin plate during arterial infusion of MB. Circulating MBs were visualized with an ultrasonographic device with a linear probe. BBB permeabilization was assessed by quantifying at confocal microscopy the extravasation of FITC-albumin perfused after each treatment. US-treated brains displayed BBB permeabilization exclusively in the volume under the US beam; no significant differences were observed between brains insonated with or without the polyolefin plate. Control brains not perfused with MB did not show signs of FITC-albumin extravasation. Our preclinical study suggests that polyolefin cranial plate could be implanted as a skull replacement to maintain craniotomic windows and perform post-surgical repeated BBB opening with ultrasound guidance to deliver therapeutic agents to the central nervous system

Arterially Perfused Neurosphere-Derived Cells Distribute Outside the Ischemic Core in a Model of Transient Focal Ischemia and Reperfusion In Vitro

BACKGROUND: Treatment with neural stem cells represents a potential strategy to improve functional recovery of post-ischemic cerebral injury. The potential benefit of such treatment in acute phases of human ischemic stroke depends on the therapeutic viability of a systemic vascular delivery route. In spite of the large number of reports on the beneficial effects of intracerebral stem cells injection in experimental stroke, very few studies demonstrated the effectiveness of the systemic intravenous delivery approach. METODOLOGY/PRINCIPAL FINDINGS: We utilized a novel in vitro model of transient focal ischemia to analyze the brain distribution of neurosphere-derived cells (NCs) in the early 3 hours that follow transient occlusion of the medial cerebral artery (MCA). NCs obtained from newborn C57/BL6 mice are immature cells with self-renewal properties that could differentiate into neurons, astrocytes and oligodendrocytes. MCA occlusion for 30 minutes in the in vitro isolated guinea pig brain preparation was followed by arterial perfusion with 1x10(6) NCs charged with a green fluorescent dye, either immediately or 60 minutes after reperfusion onset. Changes in extracellular pH and K(+) concentration during and after MCAO were measured through ion-sensitive electrodes. CONCLUSION/SIGNIFICANCE: It is demonstrated that NCs injected through the vascular system do not accumulate in the ischemic core and preferentially distribute in non-ischemic areas, identified by combined electrophysiological and morphological techniques. Direct measurements of extracellular brain ions during and after MCA occlusion suggest that anoxia-induced tissue changes, such as extracellular acidosis, may prevent NCs from entering the ischemic area in our in vitro model of transitory focal ischemia and reperfusion suggesting a role played by the surrounding microenviroment in driving NCs outside the ischemic core. These findings strongly suggest that the potential beneficial effect of NCs in experimental focal brain ischemia is not strictly dependent on their homing into the ischemic region, but rather through a bystander mechanism possibly mediated by the release of neuroprotective factors in the peri-infarct region

Increased Ethanol Resistance and Consumption in Eps8 Knockout Mice Correlates with Altered Actin Dynamics

Dynamic modulation of the actin cytoskeleton is critical for synaptic plasticity, abnormalities of which are thought to contribute to mental illness and addiction. Here we report that mice lacking Eps8, a regulator of actin dynamics, are resistant to some acute intoxicating effects of ethanol and show increased ethanol consumption. In the brain, the N-methyl-Daspartate (NMDA) receptor is a major target of ethanol. We show that Eps8 is localized to postsynaptic structures and is part of the NMDA receptor complex. Moreover, in Eps8 null mice, NMDA receptor currents and their sensitivity to inhibition by ethanol are abnormal. In addition, Eps8 null neurons are resistant to the actin-remodeling activities of NMDA and ethanol. We propose that proper regulation of the actin cytoskeleton is a key determinant of cellular and behavioral responses to ethanol

Experimental protocols.

<p>Ischemia was induced for 30 minutes, 2 hours after the <i>in vitro</i> placement of the isolated brain. NC were perfused for 1 h immediately either after the reopening of the vessel or 1 our later (protocol 2). The perfusion was followed by a wash-out period with a solution without NCs. At 5 hours <i>in vitro</i> the brains were fixed for immunohistochemistry. The bottom of the panel shows an example of simultaneous DC recordings from 4 different sites in an isolated guinea pig brain. Hypoxic depressions (HD) were recorded in the electrodes located in the regions vascularized by the occluded MCA. Potentials evoked by LOT stimulation before and during the first part of ischemia (arrowhead) disappeared when HD occurred, and recovered during MCA reperfusion. Evoked potentials in the hemisphere contralateral to MCA occlusion were not altered.</p

Mean NCs density (cells/cm²) in the MAP-2 positive zone contralateral (left column) and ipsilateral (middle column) to the ischemic side and in the MAP-2 negative zone of the ischemic hemisphere (right column) in 6 different experiments.

<p>On the left, the number of slices on which counts were made for each experiments is indicated.</p

Confocal fluorescence microscope images of PECAM-1 stained sections (50 µm thick) cut from the piriform region of brains fixed at the end of the electrophysiological experiment.

<p>CFDA green fluorescent NCs and brain vessels are shown at different magnifications. Calibration bar = 100 µm. NCs were observed in close proximity to cerebral vessels.</p

Figure 2

<p>A. Recostrution of the distribution of NCs superimposed on a coronal section immunoreacted with anti-MAP-2 antibody (immunoperoxydase staining) to identify the ischemic region. Green fluorescent CFDA-stained stem cells were counted and plotted on the adjacent section stained with the MAP-2 fluorescent antibody (calibration bar = 1 cm). In B and C, microphotographs of green fluorescent CFDA-stained stem cells on sections counterstained with the fluorescent anti-MAP-2 antibody are shown at ×10 (B) and ×40 (C) magnifications. The pictures were taken in the transition region between the MAP-2⁺ and the MAP-2⁻ areas in the olfactory region. Calibration bars: 150 µm and 16 µm for B and C, respectively.</p

Figure 4

<p>(A) Changes in extracellular pH <u>(pH_e)</u> in the PC and in mOT induced by ipsilateral MCA occlusion and reperfusion. Occlusion induced a rapid metabolic acidification of the extracellular microenviroment in PC, interrupted by a transient and mild basification (arrow) associated to the hypoxic spreading depression (HD, asterisk). No changes in extracellular [H⁺] were recorded in the mOT, that is not served by the MCA. (B) Simultaneous changes in extracellular potassium concentration ([K⁺]_o)and extracellular pH in the PC after MCA occlusion and reperfusion. An initial enhancement in [K⁺] was followed by a fast and large increase in [K⁺]_o. associated to the HD. The schematic drawing on the right illustrates the position of the two-barrel recording electrodes. The field responses (FP) recorded with the conventional extracellular barrel are also shown. The period of MCA occlusion is marked by the shaded area.</p