Stimulation of the Sphenopalatine Ganglion Induces Reperfusion and Blood-Brain Barrier Protection in the Photothrombotic Stroke Model

The treatment of stroke remains a challenge. Animal studies showing that electrical stimulation of the sphenopalatine ganglion (SPG) exerts beneficial effects in the treatment of stroke have led to the initiation of clinical studies. However, the detailed effects of SPG stimulation on the injured brain are not known.The effect of acute SPG stimulation was studied by direct vascular imaging, fluorescent angiography and laser Doppler flowmetry in the sensory motor cortex of the anaesthetized rat. Focal cerebral ischemia was induced by the rose bengal (RB) photothrombosis method. In chronic experiments, SPG stimulation, starting 15 min or 24 h after photothrombosis, was given for 3 h per day on four consecutive days. Structural damage was assessed using histological and immunohistochemical methods. Cortical functions were assessed by quantitative analysis of epidural electro-corticographic (ECoG) activity continuously recorded in behaving animals.Stimulation induced intensity- and duration-dependent vasodilation and increased cerebral blood flow in both healthy and photothrombotic brains. In SPG-stimulated rats both blood brain-barrier (BBB) opening, pathological brain activity and lesion volume were attenuated compared to untreated stroke animals, with no apparent difference in the glial response surrounding the necrotic lesion.SPG-stimulation in rats induces vasodilation of cortical arterioles, partial reperfusion of the ischemic lesion, and normalization of brain functions with reduced BBB dysfunction and stroke volume. These findings support the potential therapeutic effect of SPG stimulation in focal cerebral ischemia even when applied 24 h after stroke onset and thus may extend the therapeutic window of currently administered stroke medications

Blood-Brain Barrier Dysfunction in Epileptogenesis of the Temporal Lobe

Epilepsy of the temporal lobe (TLE) is the most common form of focal epilepsy, and in adults, it most frequently develops after injury. However, the mechanisms by which a normal functioning brain turns into an epileptic one still remain obscure. Recent studies point to vascular involvement and particularly blood-brain barrier (BBB) dysfunction in the development of epilepsy. The BBB is a specialized structure which functions to control the neuronal extracellular milieu. BBB dysfunction is found in many diseases of the central nervous system, including stroke, traumatic injuries, tumors and infections. Interestingly, all these insults may initiate an epileptogenic process which eventually leads to spontaneous, recurrent seizures. This epileptogenic time frame usually lasts weeks, months, or even years in man, and days to weeks in rodents and may serve as a “window of opportunity” for the prevention of epilepsy. However, no prevention strategy exists, stressing the importance of research into the mechanisms of epileptogenesis. Here, we will underscore recent experiments suggesting that BBB dysfunction directly induces epileptogenesis. We will provide new evidence to support the hypothesis that BBB breakdown and specifically exposure of temporal lobe structures to the most common serum protein, albumin, is sufficient to induce epileptogenesis

Preferential lentiviral targeting of astrocytes in the central nervous system.

The ability to visualize and genetically manipulate specific cell populations of the central nervous system (CNS) is fundamental to a better understanding of brain functions at the cellular and molecular levels. Tools to selectively target cells of the CNS include molecular genetics, imaging, and use of transgenic animals. However, these approaches are technically challenging, time consuming, and difficult to control. Viral-mediated targeting of cells in the CNS can be highly beneficial for studying and treating neurodegenerative diseases. Yet, despite specific marking of numerous cell types in the CNS, in vivo selective targeting of astrocytes has not been optimized. In this study, preferential targeting of astrocytes in the CNS was demonstrated using engineered lentiviruses that were pseudotyped with a modified Sindbis envelope and displayed anti-GLAST IgG on their surfaces as an attachment moiety. Viral tropism for astrocytes was initially verified in vitro in primary mixed glia cultures. When injected into the brains of mice, lentiviruses that displayed GLAST IgG on their surface, exhibited preferential astrocyte targeting, compared to pseudotyped lentiviruses that did not incorporate any IgG or that expressed a control isotype IgG. Overall, this approach is highly flexible and can be exploited to selectively target astrocytes or other cell types of the CNS. As such, it can open a window to visualize and genetically manipulate astrocytes or other cells of the CNS as means of research and treatment

BBB breakdown, astroglial response and brain damage after SPG stimulation A, Two brain surface images after injection of Evans blue indicates BBB breakdown of RB-treated (left) and RB-SPG-15 min (right) rat brains.

<p>Images below display EB (blue color) intensity, color coded. Both the size of the area with increased EB and the intensity were decreased in SPG-treated rats compared to non-stimulated animals (right graph). B, Immunostaining against the astrocytic marker, GFAP (upper panels) and the microglial marker, Iba-1 (lower panel) in RB-treated (left) and the contra-lateral control hemisphere (right). Bar graphs show area measured with intracellular staining (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039636#s2" target="_blank">Methods</a>). C, Coronal sections of brains from RB (left) and RB-SPG-15 min (right) animals. Bar graphs show change in cortical volume after photothrombosis. *p<0.05.</p

Fluorescent angiography during SPG stimulation A, Two representative angiographic images of cortical surface vessels under control conditions (left) and during SPG stimulation (3 mA, 500 µs, right, white bar represents 0.1 mm).

<p>B, Intensity curves (in arbitrary intensity units, iu) for the venous (blue) and arterial (red) compartments for the venous (blue) and arterial (red) compartments (marked in (A)) during control injection (left) and SPG stimulation (right). C, % change in diameter (black) and peak-to-peak (arterial-venous) interval (red) at different stimulation intensities (1–5 mA, 500 µs).</p

A working hypothesis on SPG-induced brain protection after stroke: The ischemic core is surrounded by a peri-ischemic region which is susceptible for an irreversible injury (AKA “stroke progression”).

<p>The peri-ischemic lesion is characterized by dysfunction of the blood-brain barrier (BBB) which induces neuronal hyper-excitability, spreading depolarizations and seizures, inflammation and cellular damage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039636#pone.0039636-Shlosberg1" target="_blank">[4]</a>. SPG stimulation at an early, post-insult therapeutic time window induces vasodilation and increased rCBF, sufficient to re-perfuse the ischemic core and to reduce lesion size. When stimulation is initiated at a delayed post-insult therapeutic window (24 h), vasodilation in the peri-ischemic lesion attenuates BBB injury and the associated neuronal hyper-excitability, thus preventing progression of the primary lesion.</p

ECoG Fast Fourier Analysis: A, Averaged power of the normalized ECoG 1-3 (left) and 4–6 (right) days after photothrombosis in low and high frequency ranges (note the different right and left Y axis).

<p>B, Typical ECoG recordings of RB-treated (left) and sham animals (right). C, ECoG recording after high pass filter (71–90 Hz) of RB-treated (lower trace) and sham animals (upper trace). D, Duration of fast activity (sec/h) for the different treated groups. Symbols as in (A). *p<0.05.</p

<i>In</i><i>vivo</i> transduction with control sindMu-ZsGreen lentiviruses that display isotype IgG on their surface.

<div><p>To demonstrate specificity of the lentiviral targeting approach, <i>in </i><i>vivo</i> brain sections were transduced with sindMu-ZsGreen lentiviruses that displayed isotype IgG on their surfaces (sindMu-ZsGreen/ IgG isotype). <b>image a</b> - ZsGreen lentiviral expression; <b>image b</b> - GFAP staining for astrocytes; <b>image c</b> – NeuN staining for neurons; <b>image d</b> – merged image for ZsGreen, GFAP and NeuN expression. Indicated scale bar - 50µm(zoom x40).</p> <p><b>B</b>. Quantitation of in-vivo lentiviral targeting - Analysis of the relative percentage of ZsGreen-positive cells that also stained positively for NeuN or GFAP, following transduction with either VSV-G-ZsGreen or sindMu-ZsGreen/GLAST IgG recombinant lentiviruses. As shown, similar relative targeting index values for both astrocytes and neurons were observed when the VSV-G-ZsGreen was used. However, using sindMu-ZsGreen/GLAST IgG lentivirus, the NeuN-ZsGreen co-staining was significantly decreased, while the relative percentage of GFAP/ZsGreen-positive cells was significantly higher.</p></div