Altered function of the glutamate-aspartate transporter GLAST, a potential therapeutic target in glioblastoma.

In glioma patients, high levels of glutamate can cause brain edema and seizures. GLAST, a glutamate-aspartate transporter expressed by astrocytes with a role in glutamate uptake, is highly expressed on the plasma membrane of glioblastoma (GBM) cells, and its expression significantly correlates with shortened patient survival. Here, it was demonstrated that inhibition of GLAST expression limited the progression and invasion of GBM xenografts. Magnetic resonance spectroscopy was used to measure glutamate in GLAST-expressing gliomas showing that these tumors exhibit increased glutamate concentration compared to GLAST-depleted glioma. Despite their GLAST expression, GBM stem-like cells (GSCs) released rather than taking up glutamate due to their lack of Na+/K+-ATPase. Overexpression of Na+/K+-ATPase in these cells restored glutamate uptake and induced apoptosis. The therapeutic relevance of targeting GLAST in gliomas was assessed using the inhibitor UCPH-101. In glioma-bearing mice, a single intratumoral injection of UCPH-101 significantly increased survival by decreasing GLAST expression and inducing apoptosis. Thus, GLAST has a novel role in GBM that appears to have crucial relevance in glutamate trafficking and may thus be a new therapeutic target.

An image registration protocol to integrate electrophysiology, MRI and neuropathology data in epileptic patients explored with intracerebral electrodes.

BACKGROUND: Several attempts have been made to coregister in vivo MRI with the histopathology of surgical samples, aiming to validate new MRI biomarkers and improve the detection of epileptogenic lesions. As a further implementation, we propose a method to reconstruct the anatomical localization of the intracerebral electrodes on the histological sections, developing a coregistration protocol to match the in vivo MRI onto the ex vivo MRI obtained from the surgical specimen. NEW METHOD: Since the ex vivo MRI is natively in geometrical correspondence with histology slices, the goal of the coregistration process is to compute the transform function mapping the clinical MRI space to the ex vivo MRI. Electrodes and leads, identified in CT-MRI, can then be segmented and translated onto the histological slices. RESULTS: Step-by-step, qualitative visual inspection showed an improved matching of the anatomical structures or boundaries and electrodes positions between the two modalities. The quantitative evaluation of the coregistration protocol reported a mean error ranging between 0.82 and 1.27 mm when a sufficient number of landmarks, particularly in the core of the specimen, were clearly identified. COMPARISON WITH EXISTING METHODS: Because histology was performed according to ex vivo MRI geometry we chose to transform the in vivo onto the ex vivo MRI, differently from other methods. CONCLUSIONS: Interesting applications of the method will include correlating the locally-generated pathological electrical activity with the subtle morphological alterations of the tissue, and histologically validating the origin of signal alterations or quantitative parameter variations in MRI studies.

In vitro labelling and detection of mesenchymal stromal cells: a comparison between magnetic resonance imaging of iron-labelled cells and magnetic resonance spectroscopy of fluorine-labelled cells.

BACKGROUND: Among the various stem cell populations used for cell therapy, adult mesenchymal stromal cells (MSCs) have emerged as a major new cell technology. These cells must be tracked after transplantation to monitor their migration within the body and quantify their accumulation at the target site. This study assessed whether rat bone marrow MSCs can be labelled with superparamagnetic iron oxide (SPIO) nanoparticles and perfluorocarbon (PFC) nanoemulsion formulations without altering cell viability and compared magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) results from iron-labelled and fluorine-labelled MSCs, respectively. METHODS: Of MSCs, 2 × 106 were labelled with Molday ION Rhodamine-B (MIRB) and 2 × 106 were labelled with Cell Sense. Cell viability was evaluated by trypan blue exclusion method. Labelled MSCs were divided into four samples containing increasing cell numbers (0.125 × 106, 0.25 × 106, 0.5 × 106, 1 × 106) and scanned on a 7T MRI: for MIRB-labelled cells, phantoms and cells negative control, T1, T2 and T2* maps were acquired; for Cell Sense labelled cells, phantoms and unlabelled cells, a 19F non-localised single-pulse MRS sequence was acquired. RESULTS: In total, 86.8% and 83.6% of MIRB-labelled cells and Cell Sense-labelled cells were viable, respectively. MIRB-labelled cells were visible in all samples with different cell numbers; pellets containing 0.5 × 106 and 1 × 106 of Cell Sense-labelled cells showed a detectable 19F signal. CONCLUSIONS: Our data support the use of both types of contrast material (SPIO and PFC) for MSCs labelling, although further efforts should be dedicated to improve the efficiency of PFC labelling.

Type II focal cortical dysplasia: Ex vivo 7T magnetic resonance imaging abnormalities and histopathological comparisons.

OBJECTIVE: In the present report, the correlations between ex vivo high-resolution imaging and specific histological and ultrastructural patterns in type II focal cortical dysplasia (FCD) have been studied to explain the differences in the magnetic resonance imaging (MRI) detection of dysplasia and to contribute to the presurgical imaging evaluation of this pathology. METHODS: Surgical specimens from 13 patients with FCD IIa/b were submitted to 7T MRI scanning, and then analyzed histologically and ultrastructurally to compare the results with the MRI findings. Region of interest (ROI)-based measures on T2-weighted images (T2wi) were quantitatively evaluated in the lesion and in adjacent perilesional gray and white matter. RESULTS: Matched histological sections and 7T T2wi showed that the core of the lesion was characterized by patchy aggregates of abnormal cells and fiber disorganization related to inhomogeneity of intracortical signal intensity. The quantitative approach on T2wi can help to distinguish the lesions and perilesional areas even in a clinical MRI-negative case. The ultrastructural study showed that the strong signal hyperintensity in the white matter of FCD IIb was related to a dysmyelination process associated with severe fiber loss and abnormal cells. Less severe histopathological features were found in FCD IIa, thus reflecting their less evident MRI alterations. INTERPRETATION: We suggest that white matter abnormalities in type IIb FCD are due to defects of the myelination processes and maturation, impaired by the presence of balloon cells. To reveal the presence and the border of type II cortical dysplasia on MRI, a quantitative ROI-based analysis (coefficient of variation) is also proposed.

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.

Effective immuno-targeting of the IDH1 mutation R132H in a murine model of intracranial glioma.

The R132H mutation of cytosolic isocitrate dehydrogenase (IDH1) is present in the majority of low grade gliomas.Immunotherapy in these tumors has an interesting, still unexploited, therapeutic potential, as they are less immunosuppressive than glioblastomas. Using site-directed mutagenesis we introduced the R132H mutation into the murine glioma cell line GL261,creating mIDH1-GL261. Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant. In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261). After intracranial injection, MRI suggested that the initial growth rate was slower in mIDH1-GL261 than p-GL261 gliomas but overall survival was similar. mIDH1-GL261 gliomas showed evidence of R132H expression and of intratumoral 2HG production (evaluated by MRS and LC-MS/MS). Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF. Control mice were injected with four ovalbumin peptides or vehicle. Mice with mIDH1-GL261 but not p-GL261 gliomas treated with mIDH1 peptides survived longer than controls; 25% of them were cured. Immunized mice showed higher amounts of peripheral CD8+ T cells, higher production of IFN-γ, and evidence of anti-mIDH1 antibodies.Immunizations led to intratumoral up-regulation of IFN-γ, granzyme-b and perforin-1 and down-regulation of TGF-ß2 and IL-10. These results support the translational potential of immunotherapeutic targeting of gliomas carrying IDH1 mutations.

Potential use of human adipose mesenchymal stromal cells for intervertebral disc regeneration: a preliminary study on biglycan-deficient murine model of chronic disc degeneration.

INTRODUCTION: Biglycan is an important proteoglycan of the extracellular matrix of intervertebral disc (IVD), and its decrease with aging has been correlated with IVD degeneration. Biglycan deficient (Bgn-/0) mice lack this protein and undergo spontaneous IVD degeneration with aging, thus representing a valuable in vivo model for preliminary studies on therapies for human progressive IVD degeneration. The purpose of the present study was to assess the possible beneficial effects of adipose-derived stromal cells (ADSCs) implants in the Bgn-/0 mouse model. METHODS: To evaluate ADSC implant efficacy, Bgn-/0 mice were intradiscally (L1-L2) injected with 8x104 ADSCs at 16 months old, when mice exhibit severe and complete IVD degeneration, evident on both 7Tesla Magnetic Resonance Imaging (7TMRI) and histology. Placebo and ADSCs treated Bgn-/0 mice were assessed by 7TMRI analysis up to 12 weeks post-transplantation. Mice were then sacrificed and implanted discs were analyzed by histology and immunohistochemistry for the presence of human cells and for the expression of biglycan and aggrecan in the IVD area. RESULTS: After in vivo treatment, 7TMRI revealed evident increase in signal intensity within the discs of mice that received ADSCs, while placebo treatment did not show any variation. Ultrastructural analyses demonstrated that human ADSC survival occurred in the injected discs up to 12 weeks after implant. These cells acquired a positive expression for biglycan, and this proteoglycan was specifically localized in human cells. Moreover, ADSC treatment resulted in a significant increase of aggrecan tissue levels. CONCLUSION: Overall, this work demonstrates that ADSC implant into degenerated disc of Bgn-/0 mice ameliorates disc damage, promotes new expression of biglycan and increased levels of aggrecan. This suggests a potential benefit of ADSC implant in the treatment of chronic degenerative disc disease and prompts further studies in this field.

Immunohistochemical expression of oncological proliferation markers in the hearts of rats during normal pregnancy.

AIM: Pregnancy is characterized by left ventricular hypertrophy that is potentially accounted for by cardiomyocyte proliferation, although no such evidence is currently available. This study investigates if the left ventricular mass (LVM) increase during pregnancy implies cell hyperplasia. MATERIALS & METHODS: In nonpregnant and late-pregnant rats, cardiac function and LVM were evaluated by MRI, and cardiomyocyte dimensions and proliferations were assessed quantitatively by morphometric analysis and immunohistochemistry using oncological markers (Ki67 and MCM2). RESULTS: In late-pregnant rats, LVM and cardiomyocyte area were greater. No mitotic figures were found nor was there any significant difference between groups in Ki67 expression. MCM2 expression was related to LVM. CONCLUSION: During pregnancy, rat cardiomyocytes undergo hypertrophy but not hyperplasia; the expression of MCM2, related to LVM, suggests it could be a marker of protein synthesis. The application of oncological markers to physiological contexts may provide insight into their role within the cell cycle.

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.

Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice.

Prion diseases are caused by a conformational modification of the cellular prion protein (PrP (C)) into disease-specific forms, termed PrP (Sc), that have the ability to interact with PrP (C) promoting its conversion to PrP (Sc). In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrP (C) region involved in the interaction with PrP (Sc) thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrP (Sc) in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding.

Blurring in patients with temporal lobe epilepsy: clinical, high-field imaging and ultrastructural study.

Magnetic resonance imaging-positive temporal lobe atrophy with temporo-polar grey/white matter abnormalities (usually called 'blurring') has been frequently reported in patients with temporal lobe epilepsy associated with hippocampal sclerosis. The poor distinction of grey and white matter has been attributed to various causes, including developmental cortical abnormalities, gliosis, myelin alterations, a non-specific increase in temporal lobe water content and metabolic/perfusion alterations. However, there is still no consensus regarding the genesis of these abnormalities and no histopathological proof for a structural nature of magnetic resonance imaging changes. The aim of this study was to investigate the pathological substrate of temporo-polar blurring using different methodological approaches and evaluate the possible clinical significance of the abnormalities. The study involved 32 consecutive patients with medically intractable temporal lobe epilepsy and hippocampal sclerosis who underwent surgery after a comprehensive electroclinical and imaging evaluation. They were divided into two groups on the basis of the presence/absence of temporo-polar blurring. Surgical specimens were examined neuropathologically, and selected samples from both groups underwent high-field 7 T magnetic resonance imaging and ultrastructural studies. At the clinical level, the two groups were significantly different in terms of age at epilepsy onset (earlier in the patients with blurring) and epilepsy duration (longer in the patients with blurring). Blurring was also associated with lower neuropsychological test scores, with a significant relationship to abstract reasoning. On 7 T magnetic resonance image examination, the borders between the grey and white matter were clear in all of the samples, but only those with blurring showed a dishomogeneous signal in the white matter, with patchy areas of hyperintensity mainly in the depth of the white matter. Sections from the patients with blurring that were processed for myelin staining revealed dishomogeneous staining of the white matter, which was confirmed by analyses of the corresponding semi-thin sections. Ultrastructural examinations revealed the presence of axonal degeneration and a significant reduction in the number of axons in the patients with blurring; there were no vascular alterations in either group. These data obtained using different methodological approaches provide robust evidence that temporo-polar blurring is caused by the degeneration of fibre bundles and suggest slowly evolving chronic degeneration with the redistribution of the remaining fibres. The article also discusses the correlations between the morphological findings and clinical data.

Human skeletal muscle stem cell antiinflammatory activity ameliorates clinical outcome in amyotrophic lateral sclerosis models.

Mesenchymal stem cell (MSC) therapy is considered one of the most promising approaches for treating different neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). We previously characterized a subpopulation of human skeletal muscle-derived stem cells (SkmSCs) with MSC-like characteristics that differentiate into the neurogenic lineage in vitro. In the present study, we evaluated the SkmSC therapeutic effects in the most characterized model of spontaneous motor neuron degeneration, the Wobbler (Wr) mouse. Before evaluating the therapeutic efficacy in the Wr mouse, we followed the route of Skm-SCs at different times after intracerebroventricular injection. Two exogenous tracers, superparamagnetic iron oxide (SPIO) nanoparticles and Hoechst 33258, were used for the in vivo and ex vivo tracking of SkmSCs. We found that the loading of both Hoechst and SPIO was not toxic and efficiently labeled SkmSCs. The magnetic resonance imaging (MRI) system 7 Tesla allowed us to localize transplanted SkmSCs along the whole ventricular system up to 18 wks after injection. The ex vivo Hoechst 33258 visualization confirmed the in vivo results obtained by MRI analyses. Behavioral observations revealed a fast and sustained improvement of motor efficacy in SkmSC-treated Wr mice associated with a relevant protection of functional neuromuscular junctions. Moreover, we found that in SkmSC-treated Wr mice, a significant increase of important human antiinflammatory cytokines occurred. This evidence is in accordance with previous findings showing the bystander effect of stem cell transplantation in neurodegenerative disorders and further strengthens the hypothesis of the possible link between inflammation, cytotoxicity and ALS.