Michele Longoni Calió

Transplantation of bone marrow mesenchymal stem cells decreases oxidative stress, apoptosis, and hippocampal damage in brain of a spontaneous stroke model

Stroke is the most common cause of motor disabilities and is a major cause of mortality worldwide. Adult stem cells have been shown to be effective against neuronal degeneration through mechanisms that include both the recovery of neurotransmitter activity and a decrease in apoptosis and oxidative stress. We chose the lineage stroke-prone spontaneously hypertensive rat (SHRSP) as a model for stem cell therapy. SHRSP rats can develop such severe hypertension that they generally suffer a stroke at approximately 1 year of age. The aim of this study was to evaluate whether mesenchymal stem cells (MSCs) decrease apoptotic death and oxidative stress in existing SHRSP brain tissue. The results of qRT-PCR assays showed higher levels of the antiapoptotic Bcl-2 gene in the MSC-treated animals, compared with untreated. Our study also showed that superoxide, apoptotic cells, and by-products of lipid peroxidation decreased in MSC-treated SHRSP to levels similar those found in the animal controls, Wistar Kyoto rats. In addition, we saw a repair of morphological damage at the hippocampal region after MSC transplantation. These data suggest that MSCs have neuroprotective and antioxidant potential in stroke-prone spontaneously hypertensive rats.

Soybean concentrated extract counteracts oxidative stress in the uterus of rats.

Abstract Objective To evaluate the effects of soy isoflavone extract in the pro-oxidant/antioxidant balance in the uterus of ovariectomized rats. Methods Twenty 3-month-old adult female Wistar rats were divided into four equal groups: GI: sham-operated (estrous phase); GII: control ovariectomized rats; GIII: ovariectomized rats treated with genistein (50 μg/kg/day) by gavage; GIV: ovariectomized rats subcutaneously treated with estrogen (10 μg/kg/day). After 30 consecutive days of treatment, the rats were euthanized and the uterus removed. The distal thirds of the uterine horns were processed for histomorphometric analyses of endometrial and myometrial thicknesses and glandular area. Other regions of the uteri were kept in liquid nitrogen and subsequently processed for analysis of reactive species quantification (DCF), total antioxidant capacity (TAC) and lipid oxidation status (TBARS). Data were statistically analyzed by one-way ANOVA, complemented by the Tukey–Kramer test (p < 0.05). Results GII and GIII exhibited lower endometrial thickness, glandular area and myometrial thickness than GI and GIV, while a higher myometrial thickness was observed in GIV compared with the other groups. Moreover, the isoflavone-treated group showed lower DCF and TBARS compared to GII, and also an improvement of TAC compared to GI and GIV. Despite the significant decrease in TBARS, no significant difference in DCF nor a decrease in TAC were observed in GIV when compared to GII. Conclusion Our data show that isoflavones improve antioxidant status and counteract oxidative stress, without promoting a trophic effect in the uterus of rats.

Evaluation of the isoflavones and estrogen effects on the rat adrenal

Abstract The aim of this study was to evaluate the morphometry and the gene expression of Ki-67, VEGF and caspase 3 and the stress oxidative in the adrenal gland of ovariectomized rats treated with estrogen or isoflavones. We used 15 Wistar rats ovariectomized treated with isoflavones or estrogen during 30 days. At the end of the treatment, the left adrenal gland was removed for subsequent histological studies and the right was used to evaluate gene expression of angiogenesis (VEGF-A), cell proliferation (Ki-67), apoptose (caspase 3 clivated) and oxidative stress. Treatment with estrogen showed a largest increase in the layers of the adrenal cortex than with isoflavones. These hypertrofic effects agree with higher expression elevation of Ki67 and VEGF, which did not occur with the caspase 3, indicating that isoflavones have great proliferative effect on the adrenal gland. Similar results were also observed on superoxide quantification show that isoflavone has a protective effect against oxidative stress. Our results indicate positively the trophic therapeutic potential of isoflavones has a protective effect and can contribute to the development of effective therapies to decrease the symptoms of menopause.

POLY(LACTIC ACID) (PLA) ELECTROSPUN FIBERS IMPROVE NEUROGENESIS AND REDUCE β-AMYLOID PLAQUES IN A TRANSGENIC MOUSE MODEL OF ALZHEIMER’S DISEASE

global deterioration scale(GDS). Results: After adjusting for age, a significant difference in fibrinogen (P 1⁄4 0.045) and CRP (P 1⁄4 0.042) levels was observed among the three groups. Fibrinogen and CRP levels were higher in patients with AD than in subjects with MCI and healthy controls. Fibrinogen and CRP levels negatively and positively correlated with MMSE-K and GDS scores, respectively. Fibrinogen and CRP levels were also positively correlated. Conclusions: The present study suggests that fibrinogen and CRP levels of patients with AD might be higher than those of subjects with MCI and healthy controls. Fibrinogen and CRP levels might be negatively correlated with cognitive function. And we expect fibrinogen and CRP levels might have the role of inflammatory biomarkers for AD.

DUAL INCRETIN AGONIST REDUCES NEUROINFLAMMATION IN A TRANSGENIC MOUSE MODEL OF ALZHEIMER’S DISEASE

Background: Apolipoprotein E (APOE) is a major lipid carrier and exists in three isoforms (APOE2, E3 and E4). The main function of APOE is to transport cholesterol, phospholipids and other lipids in the form of native lipoprotein particles in the periphery and in brain. The inheritance of APOE4 allele is the major genetic risk factor for late-onset Alzheimer disease (AD), however, the mechanisms underlying this association remain elusive. While it is recognized that APOE binds to lipids in isoform-dependent manner, a comprehensive analysis of the lipid content of APOE-containing native lipoproteins in the brain is lacking. Moreover, it remains unknown if there is a differential isoform dependent role for APOE in themolecular speciation of ISF lipids and if it is related to the isoform specific impact of APOE on AD pathogenesis. We hypothesize the APOE isoform-specific effect on Ab phagocytosis is driven by the different phospholipid composition of APOE native lipid particles and/or by the differential effect of APOE alleles on the expression of immune/phagocytic receptors. Methods:LC/MS was used to determine the phospholipid composition of APOE native particles. Electron microscopy and Western blotting were used to characterize APOE-Ab complexes. We have injected APOE-Ab co-incubation mixtures into cortex of Cx3cr1, WT and TREM2-ko mice, followed by FACS sorting and RNA-seq to examine microglia transcriptome. Two-photon imaging was used to determine the dynamics of microglia surrounding the injection site. Behavior testing was performed after infusion of APOE3-Ab and APOE4-Ab complexes into the brain. Results:Lipidomic analysis demonstrated that APOE3 and APOE4 particles have a different phospholipid composition and both decreased Ab oligomer formation. Two-photon imaging showed that APOE3-Ab and APOE4-Ab complexes affected differentially microglia barrier around the injection site. Transcriptome analysis established a significant difference in the transcriptional profiling of microglia isolated from brains injected with APOE3-Ab or APOE4-Ab complexes. Finally, there was a differential behavior response toward the infusion of APOE3-Ab or APOE4-Ab in correlation with significant changes in the microglia transcriptome. Conclusions:This study demonstrates the isoform-dependent effect of APOE on microglia function and transcriptome.

Prolonged Drug-Releasing Fibers Attenuate Alzheimer’s Disease-like Pathogenesis

Delivering drugs and agents to the brain is a huge challenge, especially for chronic neurodegenerative disorders, such as Alzheimers disease (AD). For this, prolonged and sustained release methods to increase brain uptake represent an impacting concept. The bioresorbable polymer poly-lactic acid (PLA) has high potential for medical implants; at the same time, glucagon-like peptide-1 (GLP-1) analogues have considerable neuroprotective attributes and represent a therapeutic strategy for AD. Here, a biodevice is produced by electrospinning PLA with a GLP-1 analogue (liraglutide, LG), coated with a thin layer of gelatin. The biodevice is subcutaneously implanted in a transgenic mouse model of AD and LG is released for 14 days in mice serum. After 4 weeks of implantation, crucial hallmarks of the AD are highly diminished: hippocampal senile amyloid β plaque load and neuroinflammatory markers. Furthermore, neurogenesis is enhanced in the subventricular zone, an important neurogenic niche in the brain. The designed biodevice holds great promise for being an affordable candidate to act as a prolonged drug provider, promoting LG mission through increasing its lifetime, constituting a relevant approach for old and impaired brain.

NOVEL BIODEVICE RELEASES DRUG IN VIVO FOR 14 DAYS AND AVOIDS DNA DAMAGE IN STRESS-INDUCED NEUROBLASTOMA CELLS: A PROMISE FOR ALZHEIMER'S DISEASE TREATMENT

Background: PharmaCOG is an industry-academic European project aimed at identifying reliable biomarkers that are sensitive to disease progression in patients with mild cognitive impairment. Several automated methods for quantitative assessment of white matter hyperintensities (WMH) have been recently developed (for a review see Caligiuri, 2015). However, the longitudinal reproducibility of these approaches has been poorly investigated. Here we present preliminary work aimed at evaluating the across-session test-retest reproducibility of the automated WMH quantification computed with SPM12 (Schmidt, 2012) on a group of healthy elderly subjects. Methods:Eleven 3T MRI sites (Siemens, GE, Philips) participated across Italy, Spain, France, Germany, Greece and The Netherlands. The acquisition protocol includes one 2D FLAIR sequence: acceleration factor in the range of 1.5 to 2 where possible (GRAPPA, SENSE and ASSET in Siemens, Philips and GE systems, respectively), 0.9x0.9x4mm, with TE/TR/ TI as recommended by the ADNI project (Jack, 2008). Five local healthy volunteers (55-90 years) per site were scanned in two sessions a week apart. The segmentation of WMH was performed using lesion segmentation tool (LST) version 2.0.15 (Schmidt, 2012) with optimized parameters and applying the longitudinal correction. The WMH volume and number of lesions reliability for each subject was computed evaluating testretest absolute differences relative to the mean. Results: Visual assessment indicates good segmentation results. The WMH volume was comparable across sites (Kruskal–Wallis, p1⁄4.143) and was around 1.6 ml (SD1⁄42.9). The reproducibility error of total WMH volume averaged across sites was 2.3% (SD1⁄4 7.0) while that reported for the lesion number was 1.9% (SD1⁄47.0). None of the WMH volume reproducibility metrics showed MRI site effects (Kruskall-Wallis test, p>.185). Conclusions: Despite the differences of MRI scanner configurations across our 11 sites we found consistent WMH volumes and lesion count reliability. Our findings suggest that the WMH volume may be a reliable longitudinal marker in multisite studies. Pharmacog is funded by the EU-FP7 for the Innovative Medicine Initiative (grant n 115009).

Preferential Localization of Iron in The Chromatin of Fe-Enriched Cells IsLinked to DNA Cleavage Sites and Control of Carcinogenesis

DNA oxidation by oxygen-radicals generated via an iron catalyzed Fenton reaction has been extensively investigated, but little is known about iron localization in the nuclei of mammalian cells. In vitro studies showed the presence of five oxygen and one nitrogen atoms in the inner coordination sphere of the Fe(II)-DNA complex using X-ray absorption spectroscopy (XANES and EXAFS). The identification of ferritin in the nucleus of cultured cells, as well as iron-protein receptors in the nuclear membrane suggests that iron is actively transported into the nucleus. Therefore, pictures from energy-loss spectroscopic imaging (ESI) are included in this mini-review to illustrate the distribution of iron in a fibroblast cell line. These fibroblasts were iron-overloaded by being cultured in a medium containing Fe(III)-nitrilotriacetate (FeNTA). The elemental mapping of iron and phosphorus was coincident in ultrastructures and revealed a significant concentration of both in condensed chromatin; by contrast, the elemental mapping of nitrogen, used as a control, revealed a homogenous distribution across the entire cell. This observed preferential localization was surprising, considering the pro-oxidant status of iron and the importance of maintaining genome integrity. Interestingly, recent published works demonstrates that iron chelators such as genistein and daidzein, derived from soy isoflavones, can attenuate the expression of genes related to increased cancer risk and oxidative damage in the reproductive tract of female mice. In this same sense, mesenchymal stem cells also exhibit antioxidant properties by reducing superoxide, lipid peroxidation, and DNA breaks to healthful levels after transplantation into the brains of stroke prone spontaneously hypertensive rats. Therefore, we bring attention to the use of all potential antioxidants, particularly those with affinity for both iron and DNA, in therapies for several diseases.