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MicroRNAs, contained in exosomes or freely circulating in the plasma, might play a pivotal role in the infarct-sparing effect exerted by r emote limb ischemic postconditioning (RLIP). The aims of the present study were: (1) To evaluate the effect of pure exosomes isolated from plasma of animals subjected to RLIP systemically administered to ischemic rats; (2) To finely dissect exosomes content in terms of miRNAs; (3) To select those regulatory miRNAs specifically expressed in protective exosomes and to identify molecular pathways involved in their neurobeneficial effects. Circulating exosomes were isolated from blood of animals exposed to RLIP and administered to animals exposed to tMCAO by intracerebroventricular, intraperitoneal or intranasal routes. Exosomal miRNA signature was evaluated by microarray and FISH analysis. Plasmatic exosomes isolated from plasma of RLIP rats attenuated cerebral ischemia reperfusion injury and improved neurological functions until 3 days after ischemia induction. Interestingly, miR-702-3p and miR-423-5p seem to be mainly involved in exosome protective action by modulating NOD1 and NLRP3, two key triggers of neuroinflammation and neuronal death. Collectively, the results of the present work demonstrated that plasma-released exosomes after RLIP may transfer a neuroprotective signal to the brain of ischemic animals, thus representing a potentially translatable therapeutic strategy in stroke., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: REST (Repressor-Element 1 [RE1]-silencing transcription factor) inhibits Na + /Ca 2+ exchanger-1 ( Ncx1 ) transcription in neurons through the binding of RE1 site on brain promoter (Br) after stroke. We identified a new putative RE1 site in Ncx1 heart promoter (Ht) sequence ( Ht -RE1) that participates in neuronal Ncx1 transcription. Because REST recruits DNA-methyltransferase-1 (DNMT1) and MeCP2 (methyl-CpG binding protein 2) on different neuronal genes, we investigated the role of this complex in Ncx1 transcriptional regulation after stroke., Methods and Results: Luciferase experiments performed in SH-SY5Y cells demonstrated that Br activity was selectively decreased by REST, whereas Ht activity was reduced by DNMT1, MeCP2, and REST. Notably, site-direct mutagenesis of Ht- RE1 prevented REST-dependent downregulation of Ncx1 . Furthermore, in temporoparietal cortex of 8-week-old male wild-type mice (C57BL/6) subjected to transient middle cerebral artery occlusion, DNMT1, MeCP2, and REST binding to Ht promoter was increased, with a consequent DNA promoter hypermethylation. Intracerebroventricular injection of siREST prevented DNMT1/MeCP2 binding to Ht and Ncx1 downregulation, thus causing a reduction in stroke-induced damage. Consistently, in cortical neurons subjected to oxygen and glucose deprivation plus reoxygenation Ncx1 knockdown counteracted neuronal protection induced by the demethylating agent 5-azacytidine. For comparisons between 2 experimental groups, Student's t  test was used, whereas for more than 2 experimental groups, 1-way ANOVA was used, followed by Tukey or Newman Keuls. Statistical significance was set at P <0.05., Conclusions: If the results of this study are confirmed in humans, it could be asserted that DNMT1/MeCP2/REST complex disruption could be a new pharmacological strategy to reduce DNA methylation of Ht in the brain, ameliorating stroke damage.

Background: miRNA-based strategies have recently emerged as a promising therapeutic approach in several neurodegenerative diseases. Unregulated cation influx is implicated in several cellular mechanisms underlying neural cell death during ischemia. The brain constitutively active isoform of transient receptor potential melastatin 7 (TRPM7) represents a glutamate excitotoxicity-independent pathway that significantly contributes to the pathological Ca 2+ overload during ischemia., Aims: In the light of these premises, inhibition of TRPM7 may be a reasonable strategy to reduce ischemic injury. Since TRPM7 is a putative target of miRNA135a, the aim of the present paper was to evaluate the role played by miRNA135a in cerebral ischemia. Therefore, the specific objectives of the present paper were: (1) to evaluate miR135a expression in temporoparietal cortex of ischemic rats; (2) to investigate the effect of the intracerebroventricular (icv) infusion of miR135a on ischemic damage and neurological functions; and (3) to verify whether miR135a effects may be mediated by an alteration of TRPM7 expression., Methods: miR135a expression was evaluated by RT- PCR and FISH assay in temporoparietal cortex of ischemic rats. Ischemic volume and neurological functions were determined in rats subjected to transient middle cerebral artery occlusion (tMCAo) after miR135a intracerebroventricular perfusion. Target analysis was performed by Western blot., Results: Our results demonstrated that, in brain cortex, 72 h after ischemia, miR135a expression increased, while TRPM7 expression was parallelly downregulated. Interestingly, miR135a icv perfusion strongly ameliorated the ischemic damage and improved neurological functions, and downregulated TRPM7 protein levels., Conclusions: The early prevention of TRPM7 activation is protective during brain ischemia., (© 2023 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

MicroRNA (miRNA), by post-transcriptionally regulating the expression of genes involved in stroke response, represents important effectors in stroke pathophysiology. Recently, the 103/107 miRNA family emerged as a possible therapeutic target in stroke, as it controls the expression of sodium calcium exchanger 1, a plasma membrane transporter that plays a fundamental role in stroke pathophysiology. Although the neuroprotective properties of this and other miRNAs are promising, several pharmacokinetic drawbacks remain to be faced for the development of a translatable therapy based on small RNAs in CNS diseases. In the present study, to overcome these limitations, the anti-miRNA103/107 was encapsulated in specific preparations of lipid nanoparticles (LNPs), and their effectiveness was evaluated both in an in vitro model of hypoxia represented by primary neuronal cortical cultures exposed to oxygen and glucose deprivation followed by reoxygenation, and in an in vivo model of stroke obtained in rats exposed to transient occlusion of the middle cerebral artery. The results of the present study demonstrated that the encapsulation of anti-miRNA103/107 in transferrin-conjugated PEG-stabilized LNPs allowed the blood-brain barrier crossing and significantly reduced brain ischemic damage. The present achievements pave the way for the exploitation of a systemic intravenous miRNA delivery strategy in stroke therapy., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Beyond motor neuron degeneration, homozygous mutations in the survival motor neuron 1 (SMN1) gene cause multiorgan and metabolic defects in patients with spinal muscular atrophy (SMA). However, the precise biochemical features of these alterations and the age of onset in the brain and peripheral organs remain unclear. Using untargeted NMR-based metabolomics in SMA mice, we identify cerebral and hepatic abnormalities related to energy homeostasis pathways and amino acid metabolism, emerging already at postnatal day 3 (P3) in the liver. Through HPLC, we find that SMN deficiency induces a drop in cerebral norepinephrine levels in overt symptomatic SMA mice at P11, affecting the mRNA and protein expression of key genes regulating monoamine metabolism, including aromatic L-amino acid decarboxylase (AADC), dopamine beta-hydroxylase (DβH) and monoamine oxidase A (MAO-A). In support of the translational value of our preclinical observations, we also discovered that SMN upregulation increases cerebrospinal fluid norepinephrine concentration in Nusinersen-treated SMA1 patients. Our findings highlight a previously unrecognized harmful influence of low SMN levels on the expression of critical enzymes involved in monoamine metabolism, suggesting that SMN-inducing therapies may modulate catecholamine neurotransmission. These results may also be relevant for setting therapeutic approaches to counteract peripheral metabolic defects in SMA., (© 2023. The Author(s).)

Background: Amyotrophic lateral sclerosis (ALS) is a progressive and often fatal neurodegenerative disease characterized by the loss of Motor Neurons (MNs) in spinal cord, motor cortex and brainstem. Despite significant efforts in the field, the exact pathogenetic mechanisms underlying both familial and sporadic forms of ALS have not been fully elucidated, and the therapeutic possibilities are still very limited. Here we investigate the molecular mechanisms of neurodegeneration induced by chronic exposure to the environmental cyanotoxin L-BMAA, which causes a form of ALS/Parkinson's disease (PD) in several populations consuming food and/or water containing high amounts of this compound., Methods: In this effort, mice were chronically exposed to L-BMAA and analyzed at different time points to evaluate cellular and molecular alterations and behavioral deficits, performing MTT assay, immunoblot, immunofluorescence and immunohistochemistry analysis, and behavioral tests., Results: We found that cyanotoxin L-BMAA determines apoptotic cell death and a marked astrogliosis in spinal cord and motor cortex, and induces neurotoxicity by favoring TDP-43 cytoplasmic accumulation., Conclusions: Overall, our results characterize a new versatile neurotoxic animal model of ALS that may be useful for the identification of new druggable targets to develop innovative therapeutic strategies for this disease., Competing Interests: Declaration of Competing Interest All authors agree that there are no conflicts to declare. Competing interests Nothing to declare., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: The inhibition of histone deacetylase 9 (HDAC9) represents a promising druggable target for stroke intervention. Indeed, HDAC9 is overexpressed in neurons after brain ischemia where exerts a neurodetrimental role. However, mechanisms of HDAC9-dependent neuronal cell death are not yet well established. Methods: Brain ischemia was obtained in vitro by primary cortical neurons exposed to glucose deprivation plus reoxygenation (OGD/Rx) and in vivo by transient middle cerebral artery occlusion. Western blot and quantitative real-time polymerase chain reaction were used to evaluate transcript and protein levels. Chromatin immunoprecipitation was used to evaluate the binding of transcription factors to the promoter of target genes. Cell viability was measured by MTT and LDH assays. Ferroptosis was evaluated by iron overload and 4-hydroxynonenal (4-HNE) release. Results: Our results showed that HDAC9 binds to hypoxia-inducible factor 1 (HIF-1) and specificity protein 1 (Sp1), two transcription activators of transferrin 1 receptor (TfR1) and glutathione peroxidase 4 (GPX4) genes, respectively, in neuronal cells exposed to OGD/Rx. Consequently, HDAC9 induced: (1) an increase in protein level of HIF-1 by deacetylation and deubiquitination, thus promoting the transcription of the pro-ferroptotic TfR1 gene; and (2) a reduction in Sp1 protein levels by deacetylation and ubiquitination, thus resulting in a down-regulation of the anti-ferroptotic GPX4 gene. Supporting these results, the silencing of HDAC9 partially prevented either HIF-1 increase and Sp1 reduction after OGD/Rx. Interestingly, silencing of the neurodetrimental factors, HDAC9, HIF-1, or TfR1 or the overexpression of the prosurvival factors Sp1 or GPX4 significantly reduced a well-known marker of ferroptosis 4-HNE after OGD/Rx. More important, in vivo , intracerebroventricular injection of siHDAC9 reduced 4-HNE levels after stroke by preventing: (1) HIF-1 and TfR1 increase and thus the augmented intracellular iron overload; and (2) a reduction of Sp1 and its target gene GPX4. Conclusions: Collectively, results obtained suggest that HDAC9 mediates post-traslational modifications of HIF-1 and Sp1 that, in turn, increases TfR1 and decreases GPX4 expression, thus promoting neuronal ferroptosis in in vitro and in vivo models of stroke., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Introduction and Aims: The limited therapeutic options for ischemic stroke treatment render necessary the identification of new strategies. In recent years, it has been shown that natural compounds may represent a valid therapeutic opportunity. Therefore, the present study aimed to evaluate the protective effect of Ruta graveolens water extract (RGWE) in an in vivo experimental model of brain ischemia., Methods: RGWE effects on ischemic damage and neurological function were evaluated in adult rats subjected to transient occlusion of the Middle Cerebral Artery (tMCAO), receiving two intraperitoneal injections of RGWE, 100 and 300 min after the induction of ischemia. In addition, astroglial and microglial activation was measured as GFAP and IBA-1 expression by immunofluorescence and confocal microscopy analysis., Results: Treatment with RGWE containing 10 mg/kg of Rutin, the major component, ameliorates the ischemic damage and improves neurological performances. Interestingly, the pro-inflammatory states of astrocytes and microglia, respectively detected by using C3 and iNOS markers, were significantly reduced in ipsilateral cortical and striatal areas in ischemic RGWE-treated rats., Conclusions: RGWE shows a neuroprotective effect on brain infarct volume extent in a transient focal cerebral ischemia model and this effect was paralleled by the prevention of pro-inflammatory astroglial and microglial activation. Collectively, our findings support the idea that natural compounds may represent potential therapeutic opportunities against ischemic stroke., Competing Interests: Conflict of interest statement All authors agree that there are no conflicts to declare., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background and Purpose: NCX3 (Na+-Ca2+ exchanger 3) plays a relevant role in stroke; indeed its pharmacological blockade or its genetic ablation exacerbates brain ischemic damage, whereas its upregulation takes part in the neuroprotection elicited by ischemic preconditioning. To identify an effective strategy to induce an overexpression of NCX3, we examined transcription factors and epigenetic mechanisms potentially involved in NCX3 gene regulation., Methods: Brain ischemia and ischemic preconditioning were induced in vitro by exposure of cortical neurons to oxygen and glucose deprivation plus reoxygenation (OGD/Reoxy) and in vivo by transient middle cerebral artery occlusion. Western blot and quantitative real-time polymerase chain reaction were used to evaluate transcripts and proteins of GATA3 (GATA-binding protein 3), KMT2A (lysine-methyltransferase-2A), and NCX3. GATA3 and KMT2A binding on NCX3 gene was evaluated by chromatin immunoprecipitation and Rechromatin immunoprecipitation experiments., Results: Among the putative transcription factors sharing a consensus sequence on the ncx3 brain promoter region, GATA3 was the only able to up-regulate ncx3. Interestingly, GATA3 physically interacted with KMT2A, and their overexpression or knocking-down increased or downregulated NCX3 mRNA and protein, respectively. Notably, site-direct mutagenesis of GATA site on ncx3 brain promoter region counteracted GATA3 and KMT2A binding on NCX3 gene. More importantly, we found that in the perischemic cortical regions of preconditioned rats GATA3 recruited KMT2A and the complex H3K4-3me (trimethylated lysine-4 of histone-3) on ncx3 brain promoter region, thus reducing transient middle cerebral artery occlusion–induced damage. Consistently, in vivo silencing of either GATA3 or KMT2A prevented NCX3 upregulation and consequently the neuroprotective effect of preconditioning stimulus. The involvement of GATA3/KMT2A complex in neuroprotection elicited by ischemic preconditioning was further confirmed by in vitro experiments in which the knocking-down of GATA3 and KMT2A reverted the neuroprotection induced by NCX3 overexpression in cortical neurons exposed to anoxic preconditioning followed by oxygen and glucose deprivation plus reoxygenation., Conclusions: Collectively, our results revealed that GATA3/KMT2A complex epigenetically activates NCX3 gene transcription during ischemic preconditioning.

It has been demonstrated that the K + -dependent Na + /Ca 2+ exchanger, NCKX2, is a new promising stroke neuroprotective target. However, because no pharmacological activator of NCKX2 is still available, microRNA (miRNA) may represent an alternative method to modulate NCKX2 expression. In particular, by bioinformatics analysis, miR-223-5p emerged as a possible modulator of NCKX2 expression. In the light of these premises, the aims of the present study were: (1) to evaluate miR-223-5p and NCKX2 expression in the temporoparietal cortex and striatum of rats subjected to transient middle cerebral artery occlusion; (2) to evaluate whether miR-223-5p targets the 3' UTR of the NCKX2 transcript; and (3) to evaluate the effect of miR-223-5p modulation on brain ischemic volume and neurological deficits. Our results showed that miR-223-5p expression increased in a time-dependent manner in the striatum of ischemic rats in parallel with NCKX2 downregulation, and that the transfection of cortical neurons with miR-223-5p induced a reduction of NCKX2 expression. Moreover, a luciferase assay showed that miR-223-5p specifically interacts with the NCKX2 3' UTR subregion (+7037 to +8697), thus repressing NCKX2 translation. More interestingly, intracerebroventricular infusion of anti-miR-223-5p prevented NCKX2 downregulation after ischemia, thus promoting neuroprotection. The present findings support the idea that blocking miR-223-5p by antimiRNA is a reasonable strategy to reduce the neurodetrimental effect induced by NCKX2 downregulation during brain ischemia., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: In the last decades the need to find new neuroprotective targets has addressed the researchers to investigate the endogenous molecular mechanisms that brain activates when exposed to a conditioning stimulus. Indeed, conditioning is an adaptive biological process activated by those interventions able to confer resistance to a deleterious brain event through the exposure to a sub-threshold insult. Specifically, preconditioning and postconditioning are realized when the conditioning stimulus is applied before or after, respectively, the harmul ischemia., Aims and Results: The present review will describe the most common methods to induce brain conditioning, with particular regards to surgical, physical exercise, temperature-induced and pharmacological approaches. It has been well recognized that when the subliminal stimulus is delivered after the ischemic insult, the achieved neuroprotection is comparable to that observed in models of ischemic preconditioning. In addition, subjecting the brain to both preconditioning as well as postconditioning did not cause greater protection than each treatment alone., Conclusions: The last decades have provided fascinating insights into the mechanisms and potential application of strategies to induce brain conditioning. Since the identification of intrinsic cell-survival pathways should provide more direct opportunities for translational neuroprotection trials, an accurate examination of the different models of preconditioning and postconditioning is mandatory before starting any new project., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is one of the most threatening neurodegenerative disease since it causes muscular paralysis for the loss of Motor Neurons in the spinal cord, brainstem and motor cortex. Up until now, no effective pharmacological treatment is available. Two forms of ALS have been described so far: 90% of the cases presents the sporadic form (sALS) whereas the remaining 10% of the cases displays the familiar form (fALS). Approximately 20% of fALS is associated with inherited mutations in the Cu, Zn-superoxide dismutase 1 (SOD1) gene. In the last decade, ionic homeostasis dysregulation has been proposed as the main trigger of the pathological cascade that brings to motor-neurons loss. In the light of these premises, the present review will analyze the involvement in ALS pathophysiology of the most well studied metal ions, i.e., calcium, sodium, iron, copper and zinc, with particular focus to the role of ionic channels and transporters able to contribute in the regulation of ionic homeostasis, in order to propose new putative molecular targets for future therapeutic strategies to ameliorate the progression of this devastating neurodegenerative disease.

Triticum vulgare has been extensively used in traditional medicine thanks to its properties of accelerating tissue repair. The specific extract of Triticum vulgare manufactured by Farmaceutici Damor (TVE-DAMOR) is already present in some pharmaceutical formulations used in the treatment of decubitus ulcers, skin lesions and burns. It has been recently suggested that this Triticum vulgare extract may possess potential anti-inflammatory properties. In the light of these premises the aim of the present paper was to verify the anti-inflammatory role of TVE, using the LPS-stimulated microglia model of inflammation. In particular the effect of different concentrations of TVE on the release of several mediators of inflammation such as nitric oxide, IL-6, PGE2 and TNF alpha was evaluated. More important, the anti-inflammatory effect of TVE was confirmed also in primary rat microglia cultures. The results of the present study show that TVE exerts anti-inflammatory properties since it reduces the release of all the evaluated markers of inflammation, such as NO, IL6, TNF alpha and PGE2 in LPS-activated BV2 microglial cells. Intriguingly, TVE reduced microglia activation and NO release also in primary microglia. Indeed, to verify the pathway of modulation of the inflammatory markers reported above, we found that TVE restores the cytoplasmic expression of p65 protein, kwown as specific marker associated with activation of inflammatory response. The evidence for an inhibitory activity on inflammation of this specific extract of Triticum vulgare may open the way to the possibility of a therapeutical use of the Triticum vulgare extract as an anti-inflammatory compound in certain pathological states such as burns, decubitus ulcers, folliculitis and inflammation of peripheral nerve., Competing Interests: The involvement of Damor Farmaceutici in the present project does not alter our adherence to PLOS ONE policies on sharing data and materials.

Hypoxic-ischemic encephalopathy (HI) accounts for the majority of developmental, motor and cognitive deficits in children, leading to life-long neurological impairments. Since the plasmamembrane sodium/calcium exchanger (NCX) plays a fundamental role in maintaining ionic homeostasis during adult brain ischemia, in the present work we aimed to demonstrate (1)the involvement of NCX in the pathophysiology of neonatal HI and (2)a possible NCX-based pharmacological intervention. HI was induced in neonatal mice at postnatal day 7(P7) by unilateral cut of the right common carotid artery, followed by 60 min exposure to 8%O 2 . Expression profiles of NCX isoforms from embryos stage to adulthood was evaluated in the hippocampus of hypoxic-ischemic and control mice. To assess the effect of NCX pharmacological stimulation, brain infarct volume was evaluated in brain sections, obtained at several time intervals after systemic administration of the newly synthesized NCX activator neurounina. Moreover, the long term effect of NCX activation was evaluated in adult mice (P60) subjected to neonatal HI and daily treated with neurounina for three weeks. Hypoxic-ischemic insult induced a reduction of NCX1 and NCX3 expression starting from day 7 until day 60. Notably, 8 weeks after HI induction in P7 mice, NCX pharmacological stimulation not only reduced infarct volume but improved also motor behaviour, spatial and visual memory. The present study highlights the significant role of NCX in the evolution of neonatal brain injury and in the learning and memory processes that are impaired in mice injured in the neonatal period., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Preconditioning (PC) is a phenomenon wherein a mild insult induces resistance to a later, severe injury. Although PC has been extensively studied in several neurological disorders, no studies have been performed in amyotrophic lateral sclerosis (ALS). Here we hypothesize that a sub-toxic acute exposure to the cycad neurotoxin beta-methylamino-L-alanine (L-BMAA) is able to delay ALS progression in SOD1 G93A mice and that NCX3, a membrane transporter able to handle the deregulation of ionic homeostasis occurring during ALS, takes part to this neuroprotective effect. Preconditioning effect was examined on disease onset and duration, motor functions, and motor neurons in terms of functional declines and severity of histological damage in male and female mice. Our findings demonstrate that a sub-toxic dose of L-BMAA works as preconditioning stimulus and is able to delay ALS onset and to prolong ALS mice survival. Interestingly, preconditioning prevented NCX3 downregulation in SOD1 G93A mice spinal cord, leading to an increased number of motor neurons associated to a reduced astrogliosis, and reduced the denervation of neuromuscular junctions observed in SOD1 G93A mice. These protective effects were mitigated in ncx3+/- mice. This study established for the first time an animal model of preconditioning in ALS and candidates NCX3 as a new therapeutic target.

Despite the high level achieved in the field of shoulder surgery, a global consensus on rotator cuff tears management is lacking. This work is divided into two main sessions: in the first, we set questions about hot topics involved in the rotator cuff tears, from the etiopathogenesis to the surgical treatment. In the second, we answered these questions by mentioning Evidence Based Medicine. The aim of the present work is to provide easily accessible guidelines: they could be considered as recommendations for a good clinical practice developed through a process of systematic review of the literature and expert opinion, in order to improve the quality of care and rationalize the use of resources.

Methylmercury (MeHg) is a highly neurotoxic compound that, in adequate doses, can cause damage to the brain, including developmental defects and in severe cases cell death. The RE-1-silencing transcription factor (REST) has been found to be involved in the neurotoxic effects of environmental pollutants such as polychlorinated biphenyls (PCBs). In this study, we investigated the effects of MeHg treatment on REST expression and its role in MeHg-induced neurotoxicity in neuroblastoma SH-SY5Y cells. We found that MeHg exposure caused a dose- and time- dependent apoptotic cell death, as evidenced by the appearance of apoptotic hallmarks including caspase-3 processing and annexin V uptake. Moreover, MeHg increased REST gene and gene product expression. MeHg-induced apoptotic cell death was completely abolished by REST knockdown. Interestingly, MeHg (1μM/24h) increased the expression of REST Corepressor (Co-REST) and its binding with REST whereas the other REST cofactor mammalian SIN3 homolog A transcription regulator (mSin3A) was not modified. In addition, we demonstrated that the acetylation of histone protein H4 was reduced after MeHg treatment and was critical for MeHg-induced apoptosis. Accordingly, the pan-histone deacetylase inhibitor trichostatin-A (TSA) prevented MeHg-induced histone protein H4 deacetylation, thereby reverting MeHg-induced neurotoxic effect. Male mice subcutaneously injected with 10mg/kg of MeHg for 10 days showed an increase in REST expression in the granule cell layer of the cerebellum together with a decrease in histone H4 acetylation. Collectively, we demonstrated that methylmercury exposure can cause neurotoxicity by activating REST gene expression and H4 deacetylation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Homeodomain-interacting protein kinase 2 (HIPK2) is a multitalented coregulator of an increasing number of transcription factors and cofactors involved in cell death and proliferation in several organs and systems. As Hipk2(-/-) mice show behavioral abnormalities consistent with cerebellar dysfunction, we investigated whether Hipk2 is involved in these neurological symptoms. To this aim, we characterized the postnatal developmental expression profile of Hipk2 in the brain cortex, hippocampus, striatum, and cerebellum of mice by real-time PCR, western blot analysis, and immunohistochemistry. Notably, we found that whereas in the brain cortex, hippocampus, and striatum, HIPK2 expression progressively decreased with age, that is, from postnatal day 1 to adulthood, it increased in the cerebellum. Interestingly, mice lacking Hipk2 displayed atrophic lobules and a visibly smaller cerebellum than did wild-type mice. More important, the cerebellum of Hipk2(-/-) mice showed a strong reduction in cerebellar Purkinje neurons during adulthood. Such reduction is due to the activation of an apoptotic process associated with a compromised proteasomal function followed by an unpredicted accumulation of ubiquitinated proteins. In particular, Purkinje cell dysfunction was characterized by a strong accumulation of ubiquitinated β-catenin. Moreover, our behavioral tests showed that Hipk2(-/-) mice displayed muscle and balance impairment, indicative of Hipk2 involvement in cerebellar function. Taken together, these results indicate that Hipk2 exerts a relevant role in the survival of cerebellar Purkinje cells and that Hipk2 genetic ablation generates cerebellar dysfunction compatible with an ataxic-like phenotype.

Most of the current focus on developing neuroprotective therapies is aimed at preventing neuronal death. However, these approaches have not been successful despite many years of clinical trials mainly because the numerous side effects observed in humans and absent in animals used at preclinical level. Recently, the research in this field aims to overcome this problem by developing strategies which induce, mimic, or boost endogenous protective responses and thus do not interfere with physiological neurotransmission. Preconditioning is a protective strategy in which a subliminal stimulus is applied before a subsequent harmful stimulus, thus inducing a state of tolerance in which the injury inflicted by the challenge is mitigated. Tolerance may be observed in ischemia, seizure, and infection. Since it requires protein synthesis, it confers delayed and temporary neuroprotection, taking hours to develop, with a pick at 1-3 days. A new promising approach for neuroprotection derives from post-conditioning, in which neuroprotection is achieved by a modified reperfusion subsequent to a prolonged ischemic episode. Many pathways have been proposed as plausible mechanisms to explain the neuroprotection offered by preconditioning and post-conditioning. Although the mechanisms through which these two endogenous protective strategies exert their effects are not yet fully understood, recent evidence highlights that the maintenance of ionic homeostasis plays a key role in propagating these neuroprotective phenomena. The present article will review the role of protein transporters and ionic channels involved in the control of ionic homeostasis in the neuroprotective effect of ischemic preconditioning and post-conditioning in adult brain, with particular regards to the Na(+)/Ca2(+) exchangers (NCX), the plasma membrane Ca2(+)-ATPase (PMCA), the Na(+)/H(+) exchange (NHE), the Na(+)/K(+)/2Cl(-) cotransport (NKCC) and the acid-sensing cation channels (ASIC). Ischemic stroke is the third leading cause of death and disability. Up until now, all clinical trials testing potential stroke neuroprotectants failed. For this reason attention of researchers has been focusing on the identification of brain endogenous neuroprotective mechanisms activated after cerebral ischemia. In this context, ischemic preconditioning and ischemic post-conditioning represent two neuroprotecive strategies to investigate in order to identify new molecular target to reduce the ischemic damage.

Muscle injuries are frequent in athletes. Despite their high incidence, advances in clinical diagnostic criteria and imaging, their optimal management and rehabilitation strategies are still debated in literature. Furthermore, reinjury rate is high after a muscle lesion, and an improper treatment or an early return to sports can increase the rate of reinjury and complications. Most muscle injuries are managed conservatively with excellent results, and surgery is normally advocated only for larger tears. This article reviews the current literature to provide physicians and rehabilitation specialists with the necessary basic tools to diagnose, classify and to treat muscle injuries. Based on anatomy, biomechanics, and imaging features of muscle injury, the use of a recently reported new classification system is also advocated.

Muscle injuries are frequent in high demand sports. No guidelines are available in the scientific literature. ISMuLT, the "Italian Society of Muscles, Ligaments and Tendons", in line with its multidisciplinary mission, is proud to cover this gap.

Muscular architecture involves the organization of fibres in the muscle and is one of the most important factors of muscular function. Studies have demonstrated an association with muscular architecture and contraction, with an increase of the pennation angle in muscles. The aim of the study was to evaluate the change of muscular pennation angle after therapy with warm thermal water (crenotherapy)., Participants: 45 amateur athletes undertaking different sporting activities;, Group a: 30 runners;, Group B: 15 swimmers. All the athletes underwent muscular ultrasound and clinical examination before and after the 10 sessions of the thermal protocol. At baseline the groups showed different values of pennation angle (group A = 19.1° ± 3.8° vs group B = 16.7° ± 2.4°; p=0.05). Following the thermal therapy protocol, significant variation of pennation angle was detected at rest in Group A which had significantly lower values than before the treatment (17.5° ± 2.9°; p=0.01). No differences were detected in group B., Conclusions: thermal therapy induced the greatest effect on runners (Group A) as pennation angle at rest was significantly lower after the period of crenotherapy and this variation may be as a result of a smaller muscular contracture.

Background: Experimental studies suggest that mineral waters with high concentrations of calcium and bicarbonate can impact acid-base balance. The purpose of this study was to test the effect on acid-base balance and specific urine gravity, of a bicarbonate calcic mineral water (Acqua Lete®) compared to a minimally mineralized water., Methods: 88 amateur male athletes underwent two experimental trials with a modified Wingate test: the first was carried out without hydration (Control Test, Test C, n = 88); the second was carried out after one week of controlled hydration (Test with hydration, Test H, n = 88), with 1.5 L/day of a very low mineral content water (Group A, n = 44) or 1.5 L/day of Acqua Lete® (Group B, n = 44). Measure of body temperature, bioimpedance analysis, muscular ultrasound, and urinalysis were taken before (t0), immediately after (t1), 5' (t2), and 30' (t3) after exercise., Results: Hydration results in a decreased core temperature; muscular ultrasound showed increased muscle thickness after exercise related to content of body water. Regarding urinalysis, in test H, we found in both groups after exercise a significant decrease of specific urine gravity with significantly lower levels in Group B. We also found a significant increase of pH in the same Group B., Conclusions: In conclusion all the athletes hydrated with Acqua Lete® showed a positive impact on hydration status after anaerobic exercise with significant decrease of specific urine gravity and a positive effect on pH.

Unlabelled: We compared the effects of exercise on serum levels of creatin kinase (CK) in athletes with persistent hyperCKemia at rest (CK group) and in healthy athletes (control group). Prospective controlled study. Eighteen male Caucasian athletes with high serum CK levels at rest (CK between 80 and 150 U/L) and 25 male Caucasian athletes with normal serum CK levels at rest (CK between 10 and 80 U/L)., Main Outcome Measures: Blood samples were collected at rest, 30 minutes, 6 hours, 24 hours, 48 hours and 72 hours after a progressive cycloergometer test to exhaustion. The levels of serum CK and its isoenzymes were measured. In the control group, serum CK values at rest were normal (48.18 ± 14.14 U/L). After exercise, they increased slightly, though they always remained <80 U/L, decreasing to the rest level after 48 hours. The CK group had serum CK levels at rest higher than normal (116.56 ± 33.30 U/L). Serum CK levels were still outwith the normal range after 48 hours (130.11 ± 46.95 U/L) and 72 hours (116.55 ± 24.84 U/L). Serum CK levels were significantly different in both groups both before and after progressive cycloergometer test to exhaustion. In athletes with high serum CK levels at rest, serum CK levels remained elevated and had a different kinetics after exercise when compared with healthy athletes.

Muscle tissue may be damaged following intense prolonged training as a consequence of both metabolic and mechanical factors. Serum levels of skeletal muscle enzymes or proteins are markers of the functional status of muscle tissue, and vary widely in both pathological and physiological conditions. Creatine kinase, lactate dehydrogenase, aldolase, myoglobin, troponin, aspartate aminotransferase, and carbonic anhydrase CAIII are the most useful serum markers of muscle injury, but apoptosis in muscle tissues subsequent to strenuous exercise may be also triggered by increased oxidative stress. Therefore, total antioxidant status can be used to evaluate the level of stress in muscle by other markers, such as thiobarbituric acid-reactive substances, malondialdehyde, sulfhydril groups, reduced glutathione, oxidized glutathione, superoxide dismutase, catalase and others. As the various markers provide a composite picture of muscle status, we recommend using more than one to provide a better estimation of muscle stress.

AREAS OF GENERAL AGREEMENT: Total creatine kinase (CK) levels depend on age, gender, race, muscle mass, physical activity and climatic condition. High levels of serum CK in apparently healthy subjects may be correlated with physical training status, as they depend on sarcomeric damage: strenuous exercise that damages skeletal muscle cells results in increased total serum CK. The highest post-exercise serum enzyme activities are found after prolonged exercise such as ultradistance marathon running or weight-bearing exercises and downhill running, which include eccentric muscular contractions. Total serum CK activity is markedly elevated for 24 h after the exercise bout and, when patients rest, it gradually returns to basal levels. Persistently increased serum CK levels are occasionally encountered in healthy individuals and are also markedly increased in the pre-clinical stages of muscle diseases., Areas That Are Controversial: Some authors, studying subjects with high levels of CK at rest, observed that, years later, subjects developed muscle weakness and suggested that early myopathy may be asymptomatic. Others demonstrated that, in most of these patients, hyperCKemia probably does not imply disease. In many instances, the diagnosis is not formulated following routine examination with the patients at rest, as symptoms become manifest only after exercise. Some authors think that strength training seems to be safe for patients with myopathy, even though the evidence for routine exercise prescription is still insufficient. Others believe that, in these conditions, intense prolonged exercise may produce negative effects, as it does not induce the physiological muscle adaptations to physical training given the continuous loss of muscle proteins., Growing Points: High CK serum levels in athletes following absolute rest and without any further predisposing factors should prompt a full diagnostic workup with special regards to signs of muscle weakness or other simple signs that, in both athletes and sedentary subjects, are not always promptly evident. These signs may indicate subclinical muscle disease, which training loads may evidence through the onset of profound fatigue. It is probably safe to counsel athletes with suspected myopathy to continue to undertake physical activity at a lower intensity, so as to prevent muscle damage from high intensity exercise and allow ample recovery to favour adequate recovery., Areas Timely for Developing Research: CK values show great variability among individuals. Some athletes are low responders to physical training, with chronically low CK serum levels. Some athletes are high responders, with higher values of enzyme: the relationship among level of training, muscle size, fibre type and CK release after exercise should be investigated further. In addition, more details about hyperCKemia could come from the evaluation of the kinetics of CK after stress in healthy athletes with high levels of CK due to exercise, comparing the results with the ones obtained from athletes with persistent hyperCKemia at rest. Finally, it would be important to quantify the type of exercise more suited to athletes with myopathy and the intensity of exercise not dangerous for the progression of the pathology.

Aim: To study, by conventional echocardiography, left ventricular remodelling and function in master athletes, hypertension and hypertrophic cardiomyopathy., Methods: We studied 30 master athletes (MA; soccer players; mean age 43.9+/-5.9), 24 subjects with essential hypertension (HYP; 46.6+/-6), 20 patients with hypertrophic cardiomyopathy (HCM; 42.2+/-9) and 30 normal individuals (CG; 43.4+/-5). An integrated M-mode/two-dimensional echocardiographic analysis was performed to determine chambers dimensions, relative wall thickness (RWT) and left ventricular mass (LVM), indexed to height in meters raised to the power of 2.7 (LVM/h(2.7)). Cut-off levels for LVM/h(2.7) and RWT were defined to assess 4 different patterns of LV geometric remodelling. In addition, we measured indexes of global systolic performance and indexes of global diastolic function., Results: LV wall thickness and LV end-diastolic dimensions were higher in MA than controls, but significantly lower than other groups. LVH/h(2.7) was increased in 79% of HYP and in 95% of HCM, but was within the normal limits in MA. LV geometry was normal in 22 out of 30 MA (73%), while the remaining (8 athletes, 27%) showed a concentric remodelling. Systolic function (FS and EF) was normal in MA, but was slightly reduced in HYP and increased in HCM. Analysis of diastolic function showed an abnormal relaxation pattern in all HYP and 95% of HCM, but was normal in all MA. The ratio between peak filling rate and stroke volume (PFR/SV), a relatively independent index of diastolic function, was significantly greater in hypertensive patients with normal LV remodelling compared to those without it (4+/-0.39 vs. 4.91+/-0.19; P = 0.0002)., Conclusion: MA showed lower values of wall thickness, LV dimensions and LV mass compared with HYP and HCM. Despite an abnormal remodelling, all the athletes showed a normal systolic and diastolic function. The differential diagnosis between MA, HYP and HCM is feasible by accurate, comprehensive standard Doppler echocardiography.

Aim: The aim of the study was to evaluate in 263 competitive athletes possible correlations between changes induced by different sport activities in left ventricular (LV) structure and cardiac response during maximal physical effort., Methods: A total of 160 top-level endurance athletes (ATE; swimmers, runners; 28+/-4 years; 98 male) and 103 strength-trained athletes (ATS; weight-lifters, body-builders; 27+/-5 years; male), selected on the basis of training protocol (dynamic vs. static exercise), underwent standard Doppler echocardiography, heart rate variability analysis and maximal exercise stress test by bicycle ergometry. M- and B-mode echocardiographic LV measurements were determined at rest, while the following functional indexes were assessed during effort: maximal heart rate (HR), maximal systolic blood pressure (SBP) and maximal workload (Watts reached by bicycle test)., Results: The two groups were comparable for age and sex, but ATS at rest showed higher HR, SBP, and body surface area (BSA). By echo analysis, LV mass index and ejection fraction did not significantly differ between the two groups. However, ATS showed increased sum of wall thickness (septum+posterior wall), relative wall thickness and LV end-systolic stress, while LV stroke volume and LV end-diastolic diameter (P<0.01) were greater in ATE. HR variability analysis underlined in ATE increased indexes of vagal tone (P<0.01). During maximal physical effort, ATE showed a better functional capacity, with greater maximal workload (P<0.001) reached with lower maximal HR and SBP. After adjusting for HR, age, sex, BSA and SBP, distinct multiple linear regression models evidenced in ATE independent associations of maximal effort workload with LV end-diastolic diameter (P<0.001), HR (P<0.001) at rest and LV end-systolic stress (P<0.01) were found in ATE. On the other hand, independent direct correlation of SBP max during effort with sum of wall thickness (P<0.001), BSA (P<0.05) and LV end-systolic stress (P<0.001) was evidenced in ATS., Conclusions: LV structural changes in competitive athletes represent adaptation to hemodynamic overload induced by training and are consistent with different kinds of sport activity. Work capacity during exercise is positively influenced by preload increase in ATE, while increased afterload due to isometric training in ATS determines higher systemic resistance during physical effort.

The results of a radiological study carried out on a limited number of patients undergoing mitral valvuloplasty are reported. The good results achieved with this technique are confirmed objectively.