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Dorsal root ganglia (DRG) sensitive neurons represent the connection between the peripheral sensorial receptors and the central nervous system. These neurons are enwrapped individually by the satellite glial cells (SCGs) from which they receive metabolic support. Together, neuron and SCGs, become a functional unit that, in absence of the blood brain barrier, is easily exposed to external stress and damage insults. This intimate connection/relationship, both morphological and functional, can be partially pictured and studied following traditional slicing 2D histopathological techniques. Indeed, morphological cellular and subcellular alterations and changes in protein expression and/or distribution can be observed using classical techniques. However, a whole-3D approach avoids the serial sectioning required for quantitative results plus is able to show the cyto-architecture of the organ and a more complete picture of the anatomical relationship between cell populations close to physiological conditions. Here we use a 3D imaging technique to show the cyto-architecture of the DRG after “colouring” by immunofluorescence the different DRG cell populations and to assess alterations in DRG of neuropathic rats. CGRP, IB4 and MAP2 markers were useful to study the different neuronal populations. The IB4-MAP2 combination was able to label all neurons while the CGRP-IB4 couple could not but still both settings showed a small subpopulation of neurons where the proteins were co-expressed. Moreover, GFAP, ATF3 and connexin 43 were used as markers of damage in the DRG from neuropathic animals.

Neurotoxicity is the most debilitating non-haematological adverse effect of paclitaxel (PTX) in cancer patients. Symptoms are typical of a sensory peripheral neuropathy and the incidence and degree are dependent on the cumulative dose. The symptoms include paraesthesia, disaesthesia, tingling, and numbness. Moreover, many patients develop allodynia and hyperalgesia, experiencing a severe neuropathic pain (NP). NP can originate from a peripheral sensitization then transmitted to upper centers in the central nervous system where it can determine structural and functional changes (altered neuronal discharge patterns). Preliminary evidences in other NP models showed an abundant microvascular neoangiogenesis in the primary somatosensory cortex, specifically on the hindlimb projection. The aim of this work is to investigate the microstructural vascular anomalies both in the central somatosensory pathway and peripheral (dorsal root ganglia, DRG) compartments in rats exposed to chronic PTX treatment. We treated 24 rats with PTX 10 mg/kg once a week for 4 weeks to induce a painful peripheral neuropathy. Animals were tested for neurophysiological abnormalities and behavioral NP before and after treatment. Then animals were sacrificed and perfused with fixative and/or indian-ink before collecting samples. Samples have been analyzed at synchrotron radiation resources by X-ray Phase-Contrast Tomography (XPCT) Imaging (Diamond, Didcot, UK and ESRF, Grenoble, France). Volume rendering allowed for a detailed visualization of vasculature at the sub micrometric scale. Quantitative and morphological analyses of micro-vascular structures with comparative comparisons between control and NP rats. Histochemical and histological evaluations have been performed to validate the results obtained by XPCT. A significant increased number of vessels has been found in NP samples, suggesting a neoangiogenesis at the capillary level in NP condition. The effect was larger (about +173%) on central stations, still relevant (+91%) in L4-L5 spinal cord and noticeable (+56%) in related DRG. Specifical analyses indicated that neo-formed vessels were smaller than 15 micron. NP samples were accompanied by significant decrement of the number of branch points and the tortuosity, factors showed to furtherly compromise normal microcirculation and neuronal activity. These events have been confirmed by the positivity to vessel neogenesis made by tomato lectin staining in all compartments and by morphological-histological observations at light and confocal microscopy. Evidences of vascular neo-genesis at capillary level have been found in neuropathic rats treated with PTX. These findings could shed light on new pathogenetic mechanisms and potential novel therapeutic approaches.

The rapid institution of mechanical circulatory support (MCS) during cardiogenic shock secondary to severe biventricular failure is strongly recommended. Despite the introduction of less-invasive devices and adequate anticoagulation protocols, the presence of vascular complications in patients treated with MCS has not yet been eliminated. Here, the authors report a 60-year-old patient treated with the Bi-Pella approach for biventricular failure. Despite anticoagulant therapy, the patient developed a floating thrombosis in the inferior vena cava extending to the right atrium after the Impella RP removal. Considering the thrombus instability and the risk of pulmonary embolism, the patient was treated urgently for a percutaneous mechanical thrombectomy using the AngioJet thrombectomy system. The procedure was completed without intraoperative complications, and both the completion angiography and transesophageal echocardiography showed complete thrombus removal. No procedure-related complications occurred, but the patient died from progressive worsening of left ventricular failure on the 16th postoperative day. In the case of proximal extensive deep vein thrombosis with an increased risk of pulmonary embolism, the use of percutaneous mechanical thrombectomy could be a therapeutic option, even in critically ill patients, due to its minimally invasive nature and low rates of complications.

Neurotoxicity is the most debilitating non-haematological adverse effect of paclitaxel (PTX) in cancer patients that report typical symptoms of a dose cumulative sensory peripheral neuropathy with paraesthesia, disaesthesia, tingling, and numbness. Many patients develop allodynia and hyperalgesia, experiencing neuropathic pain (NP). NP can originate from a peripheral sensitization then transmitted to the central nervous system where it can determine structural and functional changes. An abundant microvascular angiogenesis was described in the primary somatosensory cortex, specifically on the hindlimb projection of rats with NP of other origin. In this work, we investigated the microstructural vascular anomalies in the central somatosensory pathway and peripheral compartments (dorsal root ganglia, DRG) in rats exposed to chronic PTX treatment. Twenty-four rats were chronically treated with PTX 10 mg/kg to induce a painful peripheral neuropathy. Animals were tested for neurophysiological abnormalities and behavioral NP and finally perfused with fixative and/or indian ink before collecting samples. Samples have been analyzed at synchrotron radiation resources by X-ray Phase-Contrast Tomography (XPCT) Imaging (Diamond, Didcot, UK and ESRF, Grenoble, France). Volume rendering allowed a detailed visualization of vasculature at the sub micrometric scale. We performed a quantitative and morphological analysis of micro-vascular structures in PNS and CSN of control and NP rats. Histochemical and histological evaluations validated the results obtained by XPCT. A significant increased number of vessels has been found in NP samples, suggesting an angiogenesis at the capillary level in NP condition. The effect was larger (about +173%) in somatosensory cortex, still relevant in lumbar spinal cord and noticeable in related DRG. Specific analyses indicated that neo-formed vessels were smaller than 15 micron. Moreover, a significant decrement of the number of capillary branch points and tortuosity was evident in NP samples, suggesting an impairment of the normal microcirculation and neuronal activity. These events have been confirmed both by tomato-lectin staining, that showed a vessel neogenesis in all peripheral and central compartments, and by histological observations at light microscopy. These results shed light on new pathogenic mechanisms and potential novel therapeutic approaches for PTX-induced painful peripheral neuropathy. References • Staff NP et al. Pathogenesis of paclitaxel-induced peripheral neuropathy: A current review of in vitro and in vivo findings using rodent and human model systems. Exp Neurol. 2020 Feb;324:113121. • Colleoni M., Sacerdote P. Murine models of human neuropathic pain. Bio-chim Biophys Acta. 2010 Oct;1802(10):924-33. • Del Grosso et al. Brain angiogenesis in chronic pain. Journal of Cerebral Blood Flow & Metabolism 37(1S). BRAIN & BRAIN PET 2017. Poster viewing session.

ETNK1 kinase is responsible for the phosphorylation of ethanolamine to phosphoethanolamine (P-Et) (Kennedy, 1956, J Biol Chem). Recurrent somatic mutations occurring on ETNK1 were identified in about 13% of patients affected by atypical chronic myeloid leukemia (aCML), in 3-14% of chronic myelomonocytic leukemia (CMML), and in 20% of systemic mastocytosis (SM) patients with eosinophilia (Gambacorti-Passerini, 2015, Blood; Lasho, 2015, Blood Cancer J). ETNK1 mutations, encoding for H243Y, N244S/T/K, and G245V/A amino acid substitutions, cluster in a very narrow region of the ETNK1 catalytic domain and cause an impairment of ETNK1 enzymatic activity leading to a significant decrease in the intracellular concentration of P-Et (Gambacorti-Passerini, 2015, Blood). Despite this evidence, however, their oncogenic role remained largely unexplained. Here, we investigated the specific role of these mutations by using cellular CRISPR/Cas9 and ETNK1 overexpression models as well as aCML patients' samples. We showed that mutated ETNK1 causes a significant increase in mitochondrial activity (1.87 fold increase compared to WT; p=0.0002) and in ROS production (2.05 fold increase compared to WT; p Disclosures Rea: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Gambacorti-Passerini:Bristol-Myers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.

Recurrent somatic mutations in ETNK1 (Ethanolamine-Kinase-1) were identified in several myeloid malignancies and are responsible for a reduced enzymatic activity. Here, we demonstrate in primary leukemic cells and in cell lines that mutated ETNK1 causes a significant increase in mitochondrial activity, ROS production, and Histone H2AX phosphorylation, ultimately driving the increased accumulation of new mutations. We also show that phosphoethanolamine, the metabolic product of ETNK1, negatively controls mitochondrial activity through a direct competition with succinate at mitochondrial complex II. Hence, reduced intracellular phosphoethanolamine causes mitochondria hyperactivation, ROS production, and DNA damage. Treatment with phosphoethanolamine is able to counteract complex II hyperactivation and to restore a normal phenotype., ETNK1 mutations are recurrent in leukemia but how they contribute to oncogenesis is still unclear. Here, the authors show that ETNK1 mutations increase mitochondrial activity, ROS and H2AX levels and that these effects can be rescued upon phosphoethanolamine supplementation.

X-ray free-electron lasers (FELs), which amplify light emitted by a relativistic electron beam, are extending nonlinear optical techniques to shorter wavelengths, adding element specificity by exciting and probing electronic transitions from core levels. These techniques would benefit tremendously from having a stable FEL source, generating spectrally pure and wavelength-tunable pulses. We show that such requirements can be met by operating the FEL in the so-called echo-enabled harmonic generation (EEHG) configuration. Here, two external conventional lasers are used to precisely tailor the longitudinal phase space of the electron beam before emission of X-rays. We demonstrate high-gain EEHG lasing producing stable, intense, nearly fully coherent pulses at wavelengths as short as 5.9 nm (~211 eV) at the FERMI FEL user facility. Low sensitivity to electron-beam imperfections and observation of stable, narrow-band, coherent emission down to 2.6 nm (~474 eV) make the technique a prime candidate for generating laser-like pulses in the X-ray spectral region, opening the door to multidimensional coherent spectroscopies at short wavelengths. Echo-enabled harmonic generation in a free-electron laser enables 45th harmonic pulses from a 264 nm wavelength seed, yielding 5.9 nm wavelength coherent output.

Traumatism of head arteries is rare, but among them, the superficial temporal artery is the most exposed and less protected vessel. A pseudoaneurysm of the superficial temporal artery may occur after blunt head trauma in old patients or during vigorous activity in younger people. Diagnosis should be made primarily upon history and physical examination, while duplex ultrasound is appropriate to confirm the diagnosis and CT scan to exclude other possible concomitant pathologies. Direct surgical treatment is the first and main option to solve bleeding and prevent future complications. Here reported the case of an old woman treated for a post-traumatic STA pseudoaneurysm.

Background Dense and unbiased cellular-resolution representations of extended volumetric central nervous system soft-tissue anatomy are difficult to obtain, even in experimental post-mortem settings. Interestingly, X-ray phase-contrast computed tomography (X-PCI-CT), an emerging soft-tissue-sensitive volumetric imaging technique, can provide multiscale organ- to cellular-level morphological visualizations of neuroanatomical structure. New Method Here, we tested different nervous-tissue fixation procedures, conventionally used for transmission electron microscopy, to better establish X-PCI-CT-specific sample-preparation protocols. Extracted rat spinal medullas were alternatively fixed with a standard paraformaldehyde-only aldehyde-based protocol, or in combination with glutaraldehyde. Some specimens were additionally post-fixed with osmium tetroxide. Multiscale X-PCI-CT datasets were collected at several synchrotron radiation facilities, using state-of-the-art setups with effective image voxel sizes of 3.03 to 0.33 μm3, and compared to high-field magnetic resonance imaging, histology and vascular fluorescence microscopy data. Results Multiscale X-PCI-CT of aldehyde-fixed spinal cord specimens resulted in dense histology-like volumetric representations and quantifications of extended deep spinal micro-vascular networks and of intra-medullary cell populations. Osmium post-fixation increased intra-medullary contrast between white and gray-matter tissues, and enhanced delineation of intra-medullary cellular structure, e.g. axon fibers and motor neuron perikarya. Comparison with Existing Methods Volumetric X-PCI-CT provides complementary contrast and higher spatial resolution compared to 9.4 T MRI. X-PCI-CT’s advantage over planar histology is the volumetric nature of the cellular-level data obtained, using samples much larger than those fit for volumetric vascular fluorescence microscopy. Conclusions Deliberately choosing (post-)fixation protocols tailored for optimal nervous-tissue structural preservation is of paramount importance in achieving effective and targeted neuroimaging via the X-PCI-CT technique.

BACKGROUND: The International Classification of Functioning, Disability and Health (ICF) Generic-30 Set (previously referred to as Rehabilitation Set) is a minimal set of ICF categories for reporting and assessing functioning and disability in clinical populations with different health conditions along the continuum of care. Recently, the Italian Society of Physical and Rehabilitation Medicine (SIMFER) developed an Italian modification of the simple and intuitive descriptions (SID) of these categories. This study was the first one to implement the use of the SID in practice. AIM: The main aims of this study are: 1) to implement the use of the ICF in clinical practice and research among Italian Residents in PRM, and 2) to verify if the SID made the application of ICF Generic 30 Set more user-friendly than the original descriptions; 3) to examine the prevalence of functioning problems of patients accessing Rehabilitation Services to serve as reference for the development of an ICF-based clinical data collection tool. DESIGN: Multicenter cross-sectional study. SETTING: Italian Physical Medicine and Rehabilitation (PRM) outpatient rehabilitation services. POPULATION: Patients referring to Italian PRM outpatient rehabilitation services and Italian Residents in PRM. METHODS: Each School of Specialization involved, randomly, received the ICF Generic-30 Set with the original descriptions or with the SID. Residents collected over a 4-month period (April-July 2016) patients data related to the ICF Generic-30 Set categories. Moreover, the residents self-assessed their difficulty in using the ICF Generic-30 Set with the original descriptions or with the SID, through a Numeric Rating Scale (NRS). RESULTS: Ninety-three residents collected functioning data of 864 patients (mean aged 57.7±19.3) with ICF Generic-30 Set: 304 with the original descriptions and 560 with SID. The difficulty in using the ICF Generic-30 Set with SID was rated as lower than using the original descriptions (NRS 2.8±2.5 vs. 3.5±3.1; P

Il saggio ricostruisce il coinvolgimento di Vieusseux e della Antologia nei congressi degli scienziati italiani

In this paper we introduce %CEM, a macro package allowing researchers to automatically perform coarsened exact matching (CEM) in SAS environment. CEM is a non-parametric matching method widely used by researchers to avoid the confounding influence of pre-treatment control variables to improve causal inference in quasi-experimental studies. %CEM introduces a completely automated process which allows SAS users to efficiently perform CEM in fields in which large data sets are common and where SAS is the most popular statistical tool. In addition, such a macro may be used to test several coarsening combinations of numeric variables. This option also provides a visual representation of the matching frontier, thus enabling researchers to select the optimal setting which takes into account both the L1 imbalance and the percentage of matched units. The paper concludes with an empirical application comparing computational performance and results obtained using alternative available software (SAS, R and STATA) using multiple administrative data sets from a large regional database.

Over the last few years the therapeutic approach to demyelinating diseases has radically changed, strategies having been developed aimed at partnering the classic symptomatic treatments with the most advanced regenerative medicine tools. At first, the transplantation of myelinogenic cells, Schwann cells or oligodendrocytes was suggested, but the considerable technical difficulties, (poor availability, difficulties in harvesting and culturing, and the problem of rejection in the event of non-autologous sources), shifted attention towards more versatile cellular types, such as Mesenchymal Stem Cells (MSCs). Recent studies have already demonstrate both in vitro and in vivo that glially-primed MSCs (through exposure to chemical cocktails) have myelogenic abilities. In spite of a large number of papers on glially-differentiated MSCs, little is known about the ability of undifferentiated MSCs to myelinate axons and processes. Here we have demonstrated that also undifferentiated MSCs have the ability to myelinate, since they induce the myelination of rat DRG neuron processes after direct co-culturing. In this process a pivotal role is performed by the p75 receptor. © 2013 Elsevier Inc.

Mesoporous silica materials (MSM) have been proposed as promising tools for cell specific drug delivery or fluorescent cell tracking. In cancer therapy there is an urgent need to develop a cancer cell specific drug carrier able to limit the non-specific uptake of the drug by normal cells thereby reducing serious side effects. Chemotherapy induced peripheral neurotoxicity (CIPN) is one of the most clinically relevant side effects linked to the use of several antineoplastic drugs. In this study we showed that the uptake of MSM (synthesized using a PEG surfactant-based interfacial synthesis procedure), functionalised with folic acid (MSM-FOL) after 1, 6 and 24 hours is very limited in neuronal-like cellular systems such as differentiated SH-SY5Y human neuroblastoma cells and rat embryonic dorsal root ganglia sensory neurons. By contrast, the nanoparticles are highly internalized in A549 and IGROV-1 cancer cells. The 6 hour-treatment of A549 and IGROV-1 cells with nanoparticles loaded with the antineoplastic drug cisplatin (CP) induced significant cytotoxicity with respect to CP alone. These results were observed treating IGROV-1 cells with 25 and 50 μg/ml nanoparticles doses (corresponding respectively to CP 6.25 and 12.5 μM) and treating A549 with 50 μg/ml.Our results demonstrated a selective uptake of functionalized MSM suggesting them as promising tools for targeted antineoplastic therapy. Further studies will be necessary in order to confirm if this approach may be useful in reducing neurotocity of anticancer drugs.

Human serum albumin (HSA) is the most abundant plasma protein, endowed with several biological properties unrelated to its oncotic power, such as antioxidant and free-radicals scavenging activities, binding and transport of many endogenous and exogenous substances, and regulation of endothelial function and inflammatory response. These non-oncotic activities are closely connected to the peculiarly dynamic structure of the albumin molecule. HSA undergoes spontaneous structural modifications, mainly by reaction with oxidants and saccharides; however, patients with cirrhosis show extensive post-transcriptional changes at several molecular sites of HSA, the degree of which parallels the severity of the disease. The present work reports the development and application of an innovative LC-MS analytical method for a rapid and reproducible determination of the relative abundance of HSA isoforms in plasma samples from alcoholic hepatitis (AH) patients. A condition of severe oxidative stress, similar to that observed in AH patients, is associated with profound changes in circulating HSA microheterogeneity. More interestingly, the high resolution provided by the analytical platform allowed the monitoring of novel oxidative products of HSA never reported before.

This study is aimed at describing the changes occurring in the entire peripheral nervous system sensory pathway along a 2-year observation period in a cohort of C57BL/6 mice. The neurophysiological studies evidenced significant differences in the selected time points corresponding to childhood, young adulthood, adulthood, and aging (i.e., 1, 7, 15, and 25 months of age), with a parabolic course as function of time. The pathological assessment allowed to demonstrate signs of age-related changes since the age of 7 months, with a remarkable increase in both peripheral nerves and dorsal root ganglia at the subsequent time points. These changes were mainly in the myelin sheaths, as also confirmed by the Rotating-Polarization Coherent-Anti-stokes-Raman-scattering microscopy analysis. Evident changes were also present at the morphometric analysis performed on the peripheral nerves, dorsal root ganglia neurons, and skin biopsies. This extensive, multimodal characterization of the peripheral nervous system changes in aging provides the background for future mechanistic studies allowing the selection of the most appropriate time points and readouts according to the investigation aims.

Cisplatin is one of the most effective metal-based anticancer agents, targeting a large number of solid tumours. Despite its efficacy, cisplatin treatment is still limited by severe side effects such as neuro-, hepato- and nephro-toxicity and by resistance phenomena, only partially overcome by the use of new platinum drugs (i.e. oxaliplatin and carboplatin). These problems have stimulated the research and development of alternative therapeutic strategies based on different heavy metals. In this work we investigated the in vitro activity and neurotoxicity of three anticancer complexes: [Cu(PTA)4]PF6, [Cu(thp)4]PF6 and [Au(PTA)4]PF6. Neurotoxicity was evaluated by embryonic rat dorsal root ganglia (DRG) organotypic culture model. Furthermore the extent of proteasome inhibition in rat embryonic DRG neurons was evaluated by fluorimetric assay. After 48 hours of treatment, both copper-based compounds were not neurotoxic even at higher concentrations with respect to the IC50 obtained in A549 and IGROV-1 human cancer cells while [Au(PTA)4]PF6 was neurotoxic at lower concentration than IC50 in cancer cell lines tested. Since the ubiquitin-proteasome system has been identified as molecular target in cancer cells for the heavy metal based-drug, we evaluated their ability to affect the proteasome machinery in DRG neurons. The copper-based compounds, that are not neurotoxic, do not inhibit proteasome activity in DRG neurons. On the contrary, the neurotoxic complex [Au(PTA)4] PF6, induces a significant inhibition of proteasome activity even at concentration lower than the IC50. Furthermore, based on the content in heavy metal-based atoms, the complexes used in this study are suitable candidates for Photon Activation Therapy (PAT). Pretreatment of IGROV-1 cells with [Cu(PTA)4]PF6 induces an increase cell death with respect to drug or synchrotron (SR) alone. Furthermore the SR/[Cu(PTA)4]PF6 combinational treatment induced an increase in DNA damage with respect to single treatments. Our results, together with the low IC50 of the copper compounds compared to the one observed for cisplatin, suggest them as promising compounds in anticancer treatment., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

The direct contact of Mesenchymal Stem Cells (MSCs) with Dorsal Root Ganglia sensory neurons is pivotal to prolong the neuronal survival and to support their maturation (1). Here we further investigated the mechanisms underlying this direct contact-mediated positive effect, focusing our attention on the possible interaction between MSCs and neurons, and in particular on gap junction formation. We set up direct co-cultures of MSCs and sensory neurons, and after 30 days we analyzed them. The electron microscopy analysis evidenced the presence of junctions between MSCs and neurons only in direct co-cultures. Using a diffusible dye, Calcein, we demonstrated a direct interaction among cells, with a flow of dye from MSCs to neurons. To confirm the importance of such a connection we blocked it by using a gap junction blocker, the carbenoxolone (2). The use of gap junction blocker induced a decrease of neuronal survival in co-culture, thus demonstrating the important role of gap junctions for the positive effect of MSCs. We are now investigating the possible exchanged molecules, focusing our attention on some pro-survival miRNA, such as miRNA 29b and miRNA 142-5 (3), in order to identify the molecule able to positively affect the neuronal survival., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Vesicular glutamate transporters (VGLUTs) are involved in storing glutamate for secretion at the level of glutamatergic axon terminals, and for this reason they have been extensively used as markers to identify glutamate-releasing cells. Platelets have been considered as a suitable model for studying glutamatergic dysfunction because they perform glutamate uptake and express both external transporters, and NMDA-like receptors. Here, we show that platelets express the pre-synaptic markers VGLUT1 and VGLUT2 and release glutamate following aggregation, implying a possible contributory role in the pathophysiology of stroke, migraine, and other excitotoxic disorders. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

The objective of the present study was to investigate the acute effects of alcohol on blood flow volume and velocity, on wall motion of superficial large arteries, and on systemic hemodynamics in humans. In 10 healthy volunteers small doses of alcohol were administered either orally (0.3 g/kg in 250 mL water) or intravenously (7.5 mg/kg/minute in 250 mL saline in 40 minutes). The effects of alcohol were compared with those of saline 250 mL infused for 40 minutes (6.25 mL/min). Blood velocity and systodiastolic changes of wall diameter were measured in the common carotid, femoral, and brachial arteries simultaneously with cardiac output and finger blood pressure. Skin temperature was measured at the cheek, hand, and toe. Ethanol administration caused a transitory blood pressure increase accompanied by a rise in peripheral resistances at 20 minutes. Arterial blood flow was not changed by either mode of alcohol administration at any of the measurement sites. However, this result was achieved through different compensatory mechanisms in each artery. In fact, mean carotid diameter increased after both oral and intravenous ethanol administration but remained unchanged at the brachial and femoral level. Mean blood velocity was reduced after alcohol administration at the carotid but was unchanged at the brachial and femoral level after oral or intravenous alcohol administration. Skin temperature increased 20 minutes after alcohol administration at all sites. This study shows that although acute alcohol administration does not change blood flow in superficial large arteries, it causes different autoregulatory local responses of vessel walls.

Mucopolysaccharidosis type II (MPSII or Hunter Syndrome) is a lysosomal storage disorder caused by the deficit of iduronate 2-sulfatase (IDS) activity and characterized by progressive systemic and neurological impairment. As the early mechanisms leading to neuronal degeneration remain elusive, we chose to examine the properties of neural stem cells (NSCs) isolated from an animal model of the disease in order to evaluate whether their neurogenic potential could be used to recapitulate the early phases of neurogenesis in the brain of Hunter disease patients. Experiments here reported show that NSCs derived from the subventricular zone (SVZ) of early symptomatic IDS-knockout (IDS-ko) mouse retained self-renewal capacity in vitro, but differentiated earlier than wild-type (wt) cells, displaying an evident lysosomal aggregation in oligodendroglial and astroglial cells. Consistently, the SVZ of IDS-ko mice appeared similar to the wt SVZ, whereas the cortex and striatum presented a disorganized neuronal pattern together with a significant increase of glial apoptotic cells, suggesting that glial degeneration likely precedes neuronal demise. Interestingly, a very similar pattern was observed in the brain cortex of a Hunter patient. These observations both in vitro, in our model, and in vivo suggest that IDS deficit seems to affect the late phases of neurogenesis and/or the survival of mature cells rather than NSC self-renewal. In particular, platelet-derived growth factor receptor-a-positive (PDGFR-α+) glial progenitors appeared reduced in both the IDS-ko NSCs and in the IDS-ko mouse and human Hunter brains, compared with the respective healthy controls. Treatment of mutant NSCs with IDS or PDGF throughout differentiation was able to increase the number of PDGFR-α+ cells and to reduce that of apoptotic cells to levels comparable to wt. This evidence supports IDS-ko NSCs as a reliable in vitro model of the disease, and suggests the rescue of PDGFR-α+ glial cells as a therapeutic strategy to prevent neuronal degeneration. © 2013 Macmillan Publishers Limited All rights reserved.

Purpose Alterations in mRNA for myelin proteins are reported in animal models of chemotherapy-induced peripheral neuropathies (CIPN); however, ultrastructural changes in aldehyde-fixed and plastic-embedded myelin are not evident by electron microscopy. Therefore, we used X-ray diffraction (XRD) to investigate more subtle changes in myelin sheath structure from unfixed nerves. Experimental design We used in vivo chronic animal models of CIPN in female Wistar rats, administering cisplatin (CDDP 2 mg/kg, i.p. twice/week), paclitaxel (PT 10 mg/kg, i.v. once/week) or bortezomib (0.20 mg/kg, i.v. three times/week) over a total period of 4 weeks. Animal weights were monitored, and tail nerve conduction velocity (NCV) was determined at the end of the treatments to assess the occurrence of peripheral neuropathy. Sciatic nerves were collected and the myelin structure was analyzed using electron microscopy (EM) and XRD. Results All the rats treated with the chemotherapy agents developed peripheral neuropathy, as indicated by a decrease in NCV values; however, light and electron microscopy indicated no severe pathological alterations of the myelin morphology. XRD also did not demonstrate significant differences between sciatic nerves in treated vs. control rats with respect to myelin period, relative amount of myelin, membrane structure, and regularity of membrane packing. Conclusions These results indicate that experimental peripheral neuropathy caused by CDDP, PT, and bortezomib—which are among the most widely used chemotherapy agents—does not significantly affect the structure of internodal myelin in peripheral nerve.

Bortezomib (BTZ) is a highly effective and widely used antineoplastic drug for the treatment of multiple myeloma. Despite its effectiveness, the use of this proteasome inhibitor is limited by its toxicity and in particular peripheral neurotoxicity is a major and clinically-relevant problem. In fact, since the earliest Phase I and Phase II studies, in a significant proportion of BTZ-treated patients a severe sensory peripheral neuropathy emerged as a dose-limiting side effect. Unfortunately, till now the management of BTZ-peripheral neurotoxicity is an unsolved issue. The use of animal models is one of the most widely used to investigate the neurotoxicity of anticancer drugs, provided they are able to reproduce the clinical use of the drug. Since it has been suggested that bortezomib peripheral neurotoxicity is more severe in myeloma patients that in those affected by solid cancers, the currently available animal models can be less reliable in the attempt to reproduce the clinical use of the drug. Therefore, we developed a myeloma-bearing animal model in which immunodeficient scid mice were intravenously treated with BTZ 1 mg/kg weekly for 5 weeks (q7dx5). Mice were s.c. injected into the right flank with 10x106 cell/0.1 mL/mouse of RPMI-8226 multiple myeloma cells. Treatments started three days after tumor injection.The results of this study evidenced that the administration of BTZ at a dose able to treat experimental myeloma and to induce proteasome activity inhibition comparable to that achieved in myeloma patients induced significant neurophysiological changes vs. vehicle-treated animals in conduction velocity in the caudal and digital nerves and in potential amplitude in the digital nerve. Moreover, the administration of BTZ induced evident pathological changes mostly due to axonopathy vs. vehicle-treated animals in caudal and sciatic nerve as well as in dorsal root ganglia (DRG). Therefore, this model able to mimic the clinical use of BTZ can be reliable and used to test neuroprotective strategies aimed at the reduction of BTZ peripheral neurotoxicity and to rule out their interference with BTZ anti-myeloma activity.

The positive effect of adult undifferentiated mesenchymal stem cells (MSCs) on neuronal survival has already been reported, although the mechanisms by which MSCs exert their effect are still a matter of debate. Here we have demonstrated that MSCs are able to prolong the survival of dorsal root ganglion (DRG) neurons mainly by inhibiting some proteolytic enzymes, and in particular the pathway of metalloproteinases (MMPs), a family of proteins that are involved in many neuronal processes, including survival. The inhibition of MMPs was both direct, by acting on MT-MMP1, and indirect, by acting on those proteins that regulate MMPs' activation, such as Timp-1 and Sparc. The importance of the MMPs' down-regulation for neuronal survival was also demonstrated by using N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycyl hydroxamic acid (NNGH), a wide range inhibitor of metalloproteinases, which was able to increase the survival of DRG neurons in a significant manner. The down-regulation of MMPs, obtained both by MSC contact and by chemical inhibition, led to the inactivation of caspase 3, the executor of apoptotic death in DRG neurons cultured alone, while caspase 7 was found to be irrelevant for the apoptotic process. The capacity of MSCs to prevent apoptosis mainly by inactivating the metalloproteinase pathway is an important finding that sheds light on MSCs' mechanism of action, making undifferentiated MSCs a promising tool for the treatment of many different neurodegenerative pathologies.

Transitional cell carcinoma (TCC) is the most common type of bladder cancer. Emerging evidence has suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells, the cancer stem-like cells (CSCs) or tumor initiating cells. We report on the isolation and biological characterization of putative bladder CSC populations from primary TCCs. Isolated cells were induced to proliferate in stem cell culture conditions (serum-free medium containing mitogenic growth factors). The proliferating cells formed spheroids (urospheres) and their abilities for extensive proliferation and self-renewal were assayed. Their positivity for several stem cell markers (CD133, Oct-3/4, nestin, and cytokeratins) was also assessed by immunofluorescence tests and they could have the potential to differentiate in the presence of serum. In stem cell culture conditions they gradually showed loss of proliferation, adherence to the substrate, and morphological changes, which might reflect their progressive acquisition of differentiative capacity and loss of self-renewal ability. To evaluate if effective cell selection occurred after isolation, conventional cytogenetic studies on fresh chromosome spreads immediately after isolation and after culture were carried out. In addition, a molecular cytogenetic study by UroVysion assay was carried out on paraffin-embedded tissue sections and on fresh and after culture nuclei preparations. The data collected indicated important karyotype changes and a positive selection for hypo- or near-diploid cells, losing the complexity present in fresh tumors.

Magnetic iron oxide nanoparticles (NPs) are considered for various diagnostic and therapeutic applications in brain including their use as contrast agent for magnetic resonance imaging. In delivery application, the critical step is the transport across cell layers and the internalization of NPs into specific cells, a process often limited by poor targeting specificity and low internalization efficiency. The development of the models of brain endothelial cells and choroidal plexus epithelial cells in culture has allowed us to investigate into these mechanisms. Our strategy is aimed at exploring different routes to the entrapment of iron oxide NPs in these brain related cells. Here we demonstrated that not only cells endowed with a good phagocytic activity like activated macrophages but also endothelial brain capillary and choroidal plexus epithelial cells do internalize iron oxide NPs. Our study of the intracellular trafficking of NPs by TEM, and confocal microscopy revealed that NPs are mainly internalized by the endocytic pathway. Iron oxide NPs were dispersed in water and coated with 3,4-dihydroxyl-L-phenylalanine (L-DOPA) using standard procedures. Magnetic lipid NPs were prepared by NANOVECTOR: water in oil in water (W/O/W) microemulsion process has been applied to directly coat different iron based NPs by lipid layer or to encapsulate them into Solid Lipid Nanoparticles (SLNs). By these coating/loading the colloidal stability was improved without strong alteration of the particle size distribution. Magnetic lipid NPs could be reconstituted after freeze drying without appreciable changes in stability. L-DOPA coated NPs are stable in PBS and in MEM (Modified Eagle Medium) medium. The magnetic properties of these NPs were not altered by the coating processes. We investigated the cellular uptake, cytotoxicity, and interaction of these NPs with rat brain capillary endothelial (REB4) and choroidal plexus epithelial (Z310) cells. By means of widefield, confocal microscopy and flow cytometry we studied the cell uptake of magnetic SLNs derivatized with a fluorescent reporter molecule and of L-DOPA-TRITC coated NPs. Inhibition of the caveolae-mediated pathway by preincubation with filipin and nystatin did not modify the cellular uptake of these NPs in both cell lines. Furthermore a mild decrease of the NPs cell uptake was obtained after chlorpromazine and NaN3 pretreatment, which interferes with clathrin and energy-dependent endocytosis, and cytochalasin and amiloride pretreatment which interfere with macropinocytosis. NPs particle size as such can strongly affect the efficiency of cellular uptake and the mode of endocytosis. Considering that our L-DOPA and magnetic SLNs display a medium hydrodynamic size of 120 nm with a polydispersity index of 0.3, we can assume that the cell uptake process of these NPs may develop, depending the particle size, both via clathrin mediated endocytosis and macropinocytosis and only to less extent via the pathway of caveolae-mediated endocytosis. Taken together these results let us to conclude that SLNs iron loaded and iron based L-DOPA coated NPs are internalized into brain endothelial and choroidal plexus epithelial cells and this might provide the first step of an intracellular trafficking to transport these NPs between blood and brain Refereed/Peer-reviewed

Cisplatin, paclitaxel and bortezomib belong to some of the most effective families of chemotherapy drugs for solid and haematological cancers. Epothilones represent a new family of very promising antitubulin agents. The clinical use of all these drugs is limited by their severe peripheral neurotoxicity. Several in vivo rat models have reproduced the characteristics of the peripheral neurotoxicity of these drugs. However, since only a very limited number of cancer types can be studied in immunocompetent rats, these animal models do not represent an effective way to evaluate, at the same time, the antineoplastic activity and the neurotoxic effects of the anticancer compounds. In this study, we characterized the neurophysiological impairment induced by chronic chemotherapy treatment in BALB/c mice, a strain suitable for assessing the activity of anticancer treatments. At the end of a 4-week period of treatment with cisplatin, paclitaxel, epothilone-B or bortezomib, sensory and sensory/motor nerve conduction velocities (NCV) were determined in the caudal and digital nerves and dorsal root ganglia (DRG) and sciatic nerves were collected for histopathological analysis. The electrophysiological studies revealed that all the compounds caused a statistically significant reduction in the caudal NCV, while impairment of the digital NCV was less severe. This functional damage was confirmed by the histopathological observations evidencing axonal degeneration in the sciatic nerve induced by all the drugs associated with pathological changes in DRG induced only by cisplatin and bortezomib. These results confirm the possibility to use our models to combine the study of the antineoplastic activity of anticancer drugs and of their toxic effects on the peripheral nervous system in the BALB/c mouse strain.

Understanding the physiology of human neural stem cells (hNSCs) in the context of cell therapy for neurodegenerative disorders is of paramount importance, yet large-scale studies are hampered by the slow-expansion rate of these cells. To overcome this issue, we previously established immortal, non-transformed, telencephalic-diencephalic hNSCs (IhNSCs) from the fetal brain. Here, we investigated the fate of these IhNSC's immediate progeny (i.e. neural progenitors; IhNSC-Ps) upon unilateral implantation into the corpus callosum or the hippocampal fissure of adult rat brain, 3 days after global ischemic injury. One month after grafting, approximately one fifth of the IhNSC-Ps had survived and migrated through the corpus callosum, into the cortex or throughout the dentate gyrus of the hippocampus. By the fourth month, they had reached the ipsilateral subventricular zone, CA1-3 hippocampal layers and the controlateral hemisphere. Notably, these results could be accomplished using transient immunosuppression, i.e administering cyclosporine for 15 days following the ischemic event. Furthermore, a concomitant reduction of reactive microglia (Iba1+ cells) and of glial, GFAP+ cells was also observed in the ipsilateral hemisphere as compared to the controlateral one. IhNSC-Ps were not tumorigenic and, upon in vivo engraftment, underwent differentiation into GFAP+ astrocytes, and b-tubulinIII+ or MAP2+ neurons, which displayed GABAergic and GLUTAmatergic markers. Electron microscopy analysis pointed to the formation of mature synaptic contacts between host and donor-derived neurons, showing the full maturation of the IhNSC-P-derived neurons and their likely functional integration into the host tissue. Thus, IhNSC-Ps possess long-term survival and engraftment capacity upon transplantation into the globally injured ischemic brain, into which they can integrate and mature into neurons, even under mild, transient immunosuppressive conditions. Most notably, transplanted IhNSC-P can significantly dampen the inflammatory response in the lesioned host brain. This work further supports hNSCs as a reliable and safe source of cells for transplantation therapy in neurodegenerative disorders. © 2010 Rota Nodari et al.

An unsolved question is how platinum derivatives used for solid cancer therapy cause peripheral neuropathy in patients and apoptosis in "in vitro" models of chemotherapy-induced peripheral neuropathy. DRG neurons from E15 rat embryos were treated with toxic doses of oxaliplatin or cisplatin. Here, the role of MAPKs in neuronal apoptosis was studied. Both oxaliplatin and cisplatin induced a dose-dependent neuronal apoptosis, modulated by the proteins of Bcl-2 family. Regarding MAPKs, platinum derivatives activated p38 while they reduced the active form and the total amount of JNK/Sapk. Both oxaliplatin and cisplatin activated ERKs at early stages, although they behaved differently at later stages. By using specific inhibitors of the various MAPKs it was demonstrated that the platinum-induced neuronal apoptosis is mediated by early p38 and ERK1/2 activation, while JNK/Sapk has a neuroprotective role. These results suggest a role for the different MAPKs in peripheral neuropathies characterized by apoptosis of DRG neurons.

Bortezomib is a proteasome inhibitor that shows a high antineoplastic effects mainly in the treatment of a multiple myeloma. Painful bortezomib-induced peripheral neuropathy represents the dose-limiting factors in its use in clinical practice. To reproduce the clinical pain symptoms, we have established an animal model of bortezomib-induced nociceptive sensory neuropathy. This study was performed by repeated administration of bortezomib in Wistar rats at doses of 0.15-0.20 mg/kg [3q7d] for eight weeks, followed-up by an observation period of 4 weeks. Bortezomib-treated rats had a reduced weight gain at the end of treatment if compared to controls, that recovered after the follow-up period. A significant decrease in the nerve conduction velocity in both doses group was observed after the treatment period. The sensory behavioural assessment, demonstrated the onset of mechanical allodynia after the eight weeks of treatment. Sciatic nerve and DRG specimens, collected at the end of treatment and after the follow-up period, were processed for light and electron microscope analysis to evaluated any pathological alteration on fibers and on dorsal root ganglions (DRG). Morphological evaluation described a dose-dependent axonopathy in treated rats’ sciatic nerves vs. control rats, involving also the unmyelinated fibers. No pathological alteration in most of DRG satellite cells or neurons was observed. This model can be useful for the study on the neurotoxicity and pain onset mechanisms related to the treatment with bortezomib. Supported by grants from the “Fondazione Banca del Monte di Lombardia” and the “Italian Ministry of Health”(Assessment and rehabilitation in patients with chemotherapy-induced peripheral neuropathy)

We have characterised for the first time the general and neurological side effects experienced when using a series of chronic non-lethal cisplatin + paclitaxel schedules in Wistar rats, selected according to our previous experience and the animals' maximum tolerated dose. At the pathological level, the use of combination schedules was definitely more toxic at the kidney and sternal bone marrow level than the single-agent schedules. At the neurophysiological examination based on the assessment of the nerve conduction velocity measurement in the tail nerve, we identified only one combination schedule that was more neurotoxic than the similar schedules based on single-agent administration. This observation was confirmed by the neuropathological examination performed on the sciatic nerve, dorsal root ganglia, ventral and dorsal roots. Our study supports the hypothesis that the general and, to a lesser extent, neurological effects of a combination of cisplatin and paclitaxel are different from those of the administration of both drugs as single agents. We believe that these models may be useful for testing neuroprotective strategies.

l-Glutamate is one of the major excitatory neurotransmitters in the mammalian central nervous system, but recently it has been shown to have a role also in the transduction of sensory input at the periphery, and in particular in the nociceptive pathway. An excess of glutamate is implicated in cases of peripheral neuropathies as well. Conventional therapeutic approaches for treating these diseases have focused on blocking glutamate receptors with small molecules or on reducing its synthesis of the receptors through the inhibition of glutamate carboxypeptidase II (GCPII), the enzyme that generates glutamate. In vivo studies have demonstrated that the pharmacological inhibition of GCPII can either prevent or treat the peripheral nerve changes in both BB/Wor and chemically induced diabetes in rats. In this study, we characterized the expression and distribution of glutamate transporters GLT1, GLAST, EAAC1 and of the enzyme GCPII in the peripheral nervous system of female Wistar rats. Immunoblotting results demonstrated that all glutamate transporters and GCPII are present in dorsal root ganglia (DRG) and the sciatic nerve. Immunofluorescence localization studies revealed that both DRG and sciatic nerves were immunopositive for all glutamate transporters and for GCPII. In DRG, satellite cells were positive for GLT1 and GCPII, whereas sensory neurons were positive for EAAC1. GLAST was localized in both neurons and satellite cells. In the sciatic nerve, GLT1 and GCPII were expressed in the cytoplasm of Schwann cells, whereas GLAST and EAAC1 stained the myelin layer. Our results give for the first time a complete characterization of the glutamate transporter system in the peripheral nervous system. Therefore, they are important both for understanding glutamatergic signalling in the PNS and for establishing new strategies to treat peripheral neuropathies.

Bortezomib is a new proteasome inhibitor with a high antitumor activity, but also with a potentially severe peripheral neurotoxicity that induces neuropathic pain in the patients treated with this drug. To establish a preclinical model and to characterize the neuropathic pain, bortezomib was administered to Wistar rats (0.20mg/kg three times weekly [3q7d] for a total of 4 weeks). At the end of the treatment period sensory nerve conduction velocity (SNCV) was determined in the tail of each animal. Sciatic nerve, DRG and spinal cord specimens were processed for light and electron microscope observations and morphometry. Plantar test and Dynamic Aesthesiometer Test (Ugo Basile instruments) were used at the end of treatment to evaluate hyperalgesia and allodynia in treated animals. Bortezomib induced a significant reduction in SNCV. Sciatic nerve and DRG examination and morphometric determinations demonstrated mild to moderate pathological changes, while the spinal cord was morphologically normal. Bortezomib-induced changes were also observed in dynamic aesthesiometer test but not in plantar test, indicating the presence of allodynia in treated animals. This model is relevant to the neuropathy induced by bortezomib in the treatment of human malignancies and it could be useful in increasing our knowledge regarding the mechanisms underlying bortezomib neurotoxicity and neuropathic pain. Supported in part by a “Fondazione Banca del Monte” unrestricted research grant