Distribution of superparamagnetic Au/Fe nanoparticles in an isolated guinea pig brain with an intact blood brain barrier.

Diagnosis and treatment of brain disorders, such as epilepsy, neurodegenerative diseases and tumors, would benefit from innovative approaches to deliver therapeutic or diagnostic compounds into the brain parenchyma, with either a homogeneous or a targeted localized distribution pattern. To assess the mechanistic aspect of penetration of nanoparticles (NPs) into the brain parenchyma, a complex, yet controlled and facilitated environment was used: the isolated guinea pig brain maintained in vitro by arterial perfusion. In this unique preparation the blood-brain barrier and the interactions between vascular and neuronal compartments are morphologically and functionally preserved. In this study, superparamagnetic Au/Fe nanoparticles (MUS:OT Au/Fe NPs), recently studied as a promising magnetic resonance T2 contrast agent with high cellular penetration, were arterially perfused into the in vitro isolated brain and showed high and homogeneous penetration through transcytosis into the brain parenchyma. Ultramicroscopy investigation of the in vitro isolated brain sections by TEM analysis of the electron-dense core of the MUS:OT Au/Fe NPs was conducted to understand NPs' brain penetration through the BBB after in vitro arterial perfusion and their distribution in the parenchyma. Our data suggest that MUS:OT Au/Fe NPs enter the brain utilizing a physiological route and therefore can be exploited as brain penetrating nanomaterials with potential contrast agent and theranostics capabilities.

Developmental expression of Kir4.1 in astrocytes and oligodendrocytes of rat somatosensory cortex and hippocampus.

Kir4.1 is the principal K(+) channel expressed in glial cells. It has been shown that it plays a fundamental role in K(+)-spatial buffering, an astrocyte-specific process where excess extracellular concentration of K(+) ions, generated by synaptic activity, is spatially redistributed to distant sites via astrocytic syncytia. Experimental and clinical evidence suggested that abnormality of Kir4.1 function in the brain is involved in different neurological diseases such as epilepsy, dysmyelination, and Huntington's disease. Although it has been shown that Kir4.1 is expressed predominantly in astrocytes in certain areas of the rat brain and its transcript is present in the rat forebrain as early as embryonic day E14, no information is available concerning the temporal sequence of Kir4.1 protein appearance during embryonic and post-natal development. Aim of this work was to study the expression pattern of Kir4.1 channel in rat somatosensory cortex and hippocampus during development and to examine its cellular localization with the glial and oligodendroglial markers S100-ß, GFAP, and Olig-2. Kir4.1 protein was detected since E20 and a gradual increase of Kir4.1 expression occurred between early postnatal period and adulthood. We showed a gradual shift in Kir4.1 subcellular localization from the soma of astrocytes to distal glial processes. Double immunofluorescence experiments confirmed the cellular localization of Kir4.1 in glial cells. Our data provide the first overview of Kir4.1 developmental expression both in the cortex and hippocampus and support the glial role of Kir4.1 in K(+) spatial buffering.

Increased pCREB expression and the spontaneous epileptiform activity in a BCNU-treated rat model of cortical dysplasia.

OBJECTIVE: Cortical dysplasias (CDs) represent a wide range of cortical abnormalities that closely correlate with intractable epilepsy. Rats prenatally exposed to 1-3-bis-chloroethyl-nitrosurea (BCNU) represent an injury-based model that reproduces many histopathologic features of human CD. Previous studies reported in vivo hyperexcitability in this model, but in vivo epileptogenicity has not been confirmed. METHODS: To determine whether cortical and hippocampal lesions lead to epileptiform discharges and/or seizures in the BCNU model, rats at three different ages (3, 5, and 9 months old) were implanted for long-term video electroencephalographic recording. At the end of the recording session, brain tissue was processed for histologic and immunohistochemical investigation including cAMP response element binding protein (CREB) phosphorylation, as a biomarker of epileptogenicity. RESULTS: BCNU-treated rats showed spontaneous epileptiform activity (67%) in the absence of a second seizure-provoking hit. Such activity originated mainly from one hippocampus and propagated to the ipsilateral neocortex. No epileptiform activity was found in age-matched control rats. The histopathologic investigation revealed that all BCNU rats with epileptiform activity showed neocortical and hippocampal abnormalities; the presence and the severity of these lesions did not correlate consistently with the propensity to generate epileptiform discharges. Epileptiform activity was found only in cortical areas of BCNU-treated rats in which a correlation between brain abnormalities and increased pCREB expression was observed. SIGNIFICANCE: This study demonstrates the in vivo occurrence of spontaneous epileptiform discharges in the BCNU model and shows that increased pCREB expression can be utilized as a reliable biomarker of epileptogenicity.

In vivo DTI tractography of the rat brain: an atlas of the main tracts in Paxinos space with histological comparison.

Diffusion tensor imaging (DTI) is a magnetic resonance modality that permits to characterize the orientation and integrity of white matter (WM). DTI-based tractography techniques, allowing the virtual reconstruction of WM tract pathways, have found wide application in preclinical neurological research. Recently, anatomically detailed rat brain atlases including DTI data were constructed from ex vivo DTI images, but tractographic atlases of normal rats in vivo are still lacking. We propose here a probabilistic tractographic atlas of the main WM tracts in the healthy rat brain based on in vivo DTI acquisition. Our study was carried out on 10 adult female Sprague-Dawley rats using a 7T preclinical scanner. The MRI protocol permitted a reliable reconstruction of the main rat brain bundles: corpus callosum, cingulum, external capsule, internal capsule, anterior commissure, optic tract. The reconstructed fibers were compared with histological data, proving the viability of in vivo DTI tractography in the rat brain with the proposed acquisition and processing protocol. All the data were registered to a rat brain template in the coordinate system of the commonly used atlas by Paxinos and Watson; then the individual tracts were binarized and averaged, obtaining a probabilistic atlas in Paxinos-Watson space of the main rat brain WM bundles. With respect to the recent high-resolution MRI atlases, the resulting tractographic atlas, available online, provides complementary information about the average anatomical position of the considered WM tracts and their variability between normal animals. Furthermore, reference values for the main DTI-derived parameters, mean diffusivity and fractional anisotropy, were provided. Both these results can be used as references in preclinical studies on pathological rat models involving potential alterations of WM.

Genesis of heterotopia in BCNU model of cortical dysplasia, detected by means of in utero electroporation.

Derangements of cortical development can cause a wide spectrum of malformations, generally termed 'cortical dysplasia' (CD), which are frequently associated with drug-resistant epilepsy and other neurological and mental disorders. 1,3-Bis-chloroethyl-nitrosurea (BCNU)-treated rats represent a model of CD due to the presence of histological alterations similar to those observed in human CD. BCNU is an alkylating agent that, administered at embryonic day 15 (E15), causes the loss of many cells destined to cortical layers; this results in cortical thinning but also in histological alterations imputable to migration defects, such as laminar disorganization and cortical and periventricular heterotopia. In the present study we investigated the genesis of heterotopia in BCNU-treated rats by labeling cortical ventricular zone (VZ) cells with a green fluorescent protein (GFP) expression vector by means of in utero electroporation. Here, we compared the migratory pattern and subsequent distribution of the GFP-labeled cells in the developing somatosensory cortex of control and BCNU-treated animals. To this aim, we investigated the expression of a panel of developmental marker genes which identified radial glia cells (Pax6), intermediate precursors cells (Tbr2), and postmitotic neurons destined to infragranular (Tbr1) or supragranular layers (Satb2). The VZ of BCNU-treated rats appeared disorganized since E18 and at E21 the embryos showed an altered migratory pattern: migration of superficial layers appeared delayed, with a number of migrating cells in the intermediate zone and some neurons destined to superficial layers arrested in the VZ, thus forming periventricular heterotopia. Moreover, neurons that reached their correct position did not extend their axons through the corpus callosum in the contralateral hemisphere as in the control, but toward the ipsilateral cingulated cortex. Our analysis sheds light on how a malformed cortex develops after a temporally discrete environmental insult.

Cytoarchitectural, behavioural and neurophysiological dysfunctions in the BCNU-treated rat model of cortical dysplasia.

Cortical dysplasias (CDs) include a spectrum of cerebral lesions resulting from cortical development abnormalities during embryogenesis that lead to cognitive disabilities and epilepsy. The experimental model of CD obtained by means of in utero administration of BCNU (1-3-bis-chloroethyl-nitrosurea) to pregnant rats on embryonic day 15 mimics the histopathological abnormalities observed in many patients. The aim of this study was to investigate the behavioural, electrophysiological and anatomical profile of BCNU-treated rats in order to determine whether cortical and hippocampal lesions can directly lead to cognitive dysfunction. The BCNU-treated rats showed impaired short-term working memory but intact long-term aversive memory, whereas their spontaneous motor activity and anxiety-like response were normal. The histopathological and immunohistochemical analyses, made after behavioural tests, revealed the disrupted integrity of neuronal populations and connecting fibres in hippocampus and prefrontal and entorhinal cortices, which are involved in memory processes. An electrophysiological evaluation of the CA1 region of in vitro hippocampal slices indicated a decrease in the efficiency of excitatory synaptic transmission and impaired paired pulse facilitation, but enhanced long-term potentiation (LTP) associated with hyperexcitability in BCNU-treated rats compared with controls. The enhanced LTP, associated with hyperexcitability, may indicate a pathological distortion of long-term plasticity. These findings suggest that prenatal developmental insults at the time of peak cortical neurogenesis can induce anatomical abnormalities associated with severe impairment of spatial working memory in adult BCNU-treated rats and may help to clarify the pathophysiological mechanisms of cognitive dysfunction that is often associated with epilepsy in patients with CD.