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Introduction: Heating tobacco products (HTPs) are advanced electronic cigarette models. Classified by the FDA as a modified-risk tobacco product and can be used as part of efforts to quit smoking. Using heat-not-burn (HnB) technology, these devices heat tobacco avoiding complete combustion. Although the levels of toxicants in the mainstream are significantly lower than those observed in tobacco smoke, some recent studies have raised concerns about potential health risks associated with their use, particularly regarding their effects on male gonadal function, which remain largely unexplored., Methods: Adult male Sprague-Dawley rats were exposed, whole body, 5 days/week for 4 weeks to HnB mainstream., Results: The expression of the cell cycle regulators Bax/Bcl-2 ratio is not affected, along with no changes in p-38. On the other hand, an increase in oxidative stress markers, including those associated with DNA damage, was observed in exposed animals, along with the induction of NF-kB dependent pro-inflammatory mediators: TNF-α, IL-1β, IL-6 and COX-2. Furthermore, inactivation of key androgenic enzymes, such as 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase, together with decreased testosterone synthesis suggest a potential impairment of male gonadal function., Discussion: The results indicate that animals exposed to HnB smoke show higher levels of oxidative stress markers, including those associated with DNA damage, as well as higher levels of pro-inflammatory cytokines. The impairment of some androgenic key enzymes and those related to the activity of seminiferous epithelium, together with the decrease in testosterone levels, suggest an impairment of gonadal function through the alteration of some cellular pathways typically associated with tobacco consumption., Competing Interests: Author IF was employed by Gruppo CSA—S.p.A. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Granata, Morosini, Valerii, Fagiolino, Sangiorgi, Ghini, Spisni, Vivarelli, Fairclough, Paolini and Canistro.)

Objectives: The goal of this study was to assess the effect of the COVID 19 pandemic in terms of work-related stress on a sample of 424 Italian dental hygienists (rate response 98%). It was tested which factors may influence most the perception of stress among Italian dental hygienists, which coping strategies hygienists adopted, and which protective factors may help them to cope with stress., Methods: Data were collected during a phase of moderate improvement of epidemiological conditions in Italy (Summer 2021-from June to September) through an online questionnaire, 424 questionnaires were considered for analysis., Results: Results show a perception of good satisfaction among participants about their job, although the level of stress perception seems to be significant. They have also a perception of effective risk management in the workplace. The elements that seem to influence mostly work-related stress levels are associated to (1) repetitive movements and postural changes due to the use of mechanical tools; (2) the use of personal protective equipment (e.g., surgery masks, gloves, etc.); and (3) relations with patients. Results show that participants tend to implement coping strategies such as physical activity and/or spending time with family and/or friends, but it seems that at an organisational level, no systematic measures have been taken., Conclusions: This study shows some protective factors that may help in coping with work-related stress, such as support from the dentist, a good work-life balance, role clarity with respect to the operational and professional role, the perception of self-efficacy at work, good integration into the teamwork and perceived support from colleagues., (© 2024 The Author(s). International Journal of Dental Hygiene published by John Wiley & Sons Ltd.)

Benznidazole (BNZ) serves as the primary drug for treating Chagas Disease and is listed in the WHO Model List of Essential Medicines for Children. Herein, a new child-friendly oral BNZ delivery platform is developed in the form of supramolecular eutectogels (EGs). EGs address BNZ's poor oral bioavailability and provide a flexible twice-daily dose in stick-pack format. This green and sustainable formulation strategy relies on the gelation of drug-loaded Natural Deep Eutectic Solvents (NaDES) with xanthan gum (XG) and water. Specifically, choline chloride-based NaDES form stable and biocompatible 5 mg/mL BNZ-loaded EGs. Rheological and Low-field NMR investigations indicate that EGs are viscoelastic materials comprised of two co-existing regions in the XG network generated by different crosslink distributions between the biopolymer, NaDES and water. Remarkably, the shear modulus and relaxation spectrum of EGs remain unaffected by temperature variations. Upon dilution with simulated gastrointestinal fluids, EGs results in BNZ supersaturation, serving as the primary driving force for its absorption. Interestingly, after oral administration of EGs to rats, drug bioavailability increases by 2.6-fold, with a similar increase detected in their cerebrospinal fluid. The noteworthy correlation between in vivo results and in vitro release profiles confirms the efficacy of EGs in enhancing both peripheral and central BNZ oral bioavailability., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Giorgia Nucci reports financial support was provided by Fondazione Famiglia Parmiani, Bologna. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.]., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Although the Food and Drug Administration has authorized the marketing of "heat-not-burn" (HnB) electronic cigarettes as a modified risk tobacco product (MRTP), toxicological effects of HnB smoke exposure on the brain are still unexplored. Here, paramagnetic resonance of the prefrontal cortex (PFC) of HnB-exposed rats shows a dramatic increase in reactive radical species (RRS) yield coupled with an inflammatory response mediated by NF-κB-target genes including TNF-α, IL-1β, and IL-6 and the downregulation of peroxisome proliferator-activated receptor (PPAR) alpha and gamma expression. The PFC shows higher levels of 8-hydroxyguanosine, a marker of DNA oxidative damage, along with the activation of antioxidant machinery and DNA repair systems, including xeroderma pigmentosum group C (XPC) protein complex and 8-oxoguanine DNA glycosylase 1. HnB also induces the expression of drug-metabolizing enzymes such as CYP1A1, CYP2A6, CYP2B6, and CYP2E, particularly involved in the biotransformation of nicotine and several carcinogenic agents such as aldehydes and polycyclic aromatic hydrocarbons here recorded in the HnB stick smoke. Taken together, these effects, from disruption of redox homeostasis, inflammation, PPAR manipulation along with enhanced bioactivation of neurotoxicants, and upregulation of cMYC protooncogene to impairment of primary cellular defense mechanisms, suggest a possible increased risk of brain cancer. Although the HnB device reduces the emission of tobacco toxicants, our findings indicate that its consumption may carry a risk of potential adverse health effects, especially in non-smokers so far. Further studies are needed to fully understand the long-term effects of these devices., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Vivarelli, Morosini, Rullo, Losapio, Lacorte, Sangiorgi, Ghini, Fagiolino, Franchi, Lucarini, Candeletti, Canistro, Romualdi and Paolini.)

This study investigates the correlation between the structural and release properties of solid lipid microparticles (MPs) of tristearin containing 5 % w/w of four different liquid additives used as crystal modifiers: isopropyl myristate (IM), ethyl oleate (EO), oleic acid (OA) and medium chain triglycerides (MCT). All additives accelerated the conversion of the unstable α-form of tristearin, formed after the MPs manufacturing, to the stable β-polymorph and the transformation was completed within 24 h (for IM and EO) or 48 h (for OA and MCT). The kinetic of polymorphic transition at 25 °C was investigated by simultaneous synchrotron SAXS/WAXS and DSC analysis after melting and subsequent cooling of the lipid mixture. After crystallization in the α-phase, additives accelerate the solid-solid phase transformation to β-tristearin. SAXS data showed that two types of structural modifications occurred on MPs during storage: compaction of the crystal packing (slight decrease in lamellar thickness) and crystal growth (increased number of stacked lipid lamellae). The release behavior of a model hydrophilic drug (caffeine) at two different amounts (15 % and 30 %) from MPs was studied in water and biorelevant media simulated the gastric and intestinal environment. It was particularly significant that the introduction of IM, EO and MCT were able to prolong the drug release in water, passing from a diffusion-based Higuchi kinetics to a perfect zero-order kinetic. Moreover, the overall release profiles were higher in biorelevant media, where erosion/digestion of MPs was observed. After 6 months, a moderate but statistically significant change in release profile was observed for the MPs with IM and EO, which can be correlated with the time-dependent structural alterations (i.e. larger average crystallite size) of these formulations; while MPs with OA or MCT displayed stable release profiles. These findings help to understand the correlation between release behavior, polymorphism and supramolecular-level structural modification of lipid formulations containing crystal modifiers., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The Food and Drug Administration has recently classified the IQOS electronic cigarette as a modified-risk tobacco product. However, IQOS cigarettes still release various harmful constituents typical of conventional cigarettes (CCs), although the concentrations are markedly lower. Here, we investigated the damaging effects of IQOS smoking on the liver. Male Sprague Dawley rats were exposed, whole body, 5 days/week for 4 weeks to IQOS smoke (4 sticks/day), and hepatic xenobiotic metabolism, redox homeostasis and lipidomic profile were investigated. IQOS boosted reactive radicals and generated oxidative stress. Exposure decreased cellular reserves of total glutathione (GSH) but not GSH-dependent antioxidant enzymes. Catalase and xanthine oxidase were greater in the exposed group, as were various hepatic CYP-dependent monooxygenases (CYP2B1/2, CYP1A1, CYP2A1, CYP2E1-linked). Respiratory chain activity was unaltered, while the number of liver mitochondria was increased. IQOS exposure had an impact on the hepatic lipid profile. With regard to the expression of some MAP kinases commonly activated by CC smoking, IQOS increased the p-p38/p38 ratio, while erythroid nuclear transcription factor 2 (Nrf2) was negatively affected. Our data suggest that IQOS significantly impairs liver function, supporting the precautionary stance taken by the WHO toward the use of these devices, especially by young people and pregnant women.

Background: The aim of this study was to achieve a consensus on the minimum set of outcome measures and predictors to be used in the neurosurgical practice and on the timing of outcome assessment., Methods: A consensus building approach was employed. All neurosurgical departments in Lombardy (Italy) were invited to participate by the Carlo Besta Neurologic Institute IRCCS Foundation. Three workshops were organized during which a multidisciplinary group called Neurosurgical Outcome Network (NEON) was created and the methodology to select outcome measures, predictors, and timing of outcome assessment was established. Eight working groups were created for the different neurosurgical diseases (neuro-oncological, skull base, vascular, traumatic, spinal, peripheral nervous system, malformation, functional) and 8 workshops were organized to identify the outcome measures and predictors specific for each of the neurosurgical diseases based on the experts' clinical practice and the existing literature., Results: A total of 20 neurosurgical departments participated in this study. Specific outcome measures, predictors and the timing of outcome assessment were identified for each of the 8 neurosurgical diseases. Moreover, a list of variables common to all pathologies were identified by the NEON group as further data to be collected., Conclusions: A consensus on the minimum set of outcome measures and predictors and the timing of outcome assessments for 8 neurosurgical diseases was achieved by a group of neurosurgeons of the Lombardy region, called NEON. These sets could be used in future studies for a more homogeneous data collection and as a starting point to reach further agreement also at national and international level.

Recently, Natural Deep Eutectic Solvents (NaDES) have emerged as potential solvents for boosting drug bioavailability. In this work, the mechanism of solubility enhancement of some APIs belonging to BCS class II (tolbutamide, nimesulide, domperidone and cinnarizine) in these eutectic bio-solvents was investigated in order to get deeper insights into the molecular interactions between the NaDES components and the selected drugs. Different NaDES formulations based on choline chloride, proline, solid organic acids (citric, tartaric and malic acid), sugars (glucose and xylitol) and water were prepared by mild heating (70 °C). Characterization of unloaded NaDES (pH, Karl Fisher titration, viscosity and FTIR analysis) indicated that the type of Hydrogen Bond Acceptor (HBA) and Hydrogen Bond Donor (HBD), their molar ratio as well as water amount strongly affect the extent of H-bonding interactions. Hard gelatin capsules filled with NaDES maintained their integrity until 6 months, proving that all water molecules participate in H-bond network. APIs' solubility enhancement was significant in all NaDES with respect to buffer solutions (pH 1.2 and 6.8). Analysing NaDES having Choline as HBA, it was found that the solubility of smaller molecules increased using larger HBD, while higher molecular weight APIs can be better inserted into the network formed by smaller HBD. NOE experiments demonstrated the formation of a robust supramolecular structure among the protons of choline, those of organic acid and water. In addition, 1D ROESY spectra revealed for the first time the crucial role of choline (methyl groups) in establishing hydrophobic interactions with the relative aliphatic or aromatic portion of the drugs. These data suggest the complex structure of the API-NaDES supramolecular assembly and underline that drug solubility is dependent on a balance network of H-bonds and hydrophobic interactions as well. Understanding the type of interactions between the API and NaDES is essential for their use as effective solubilisation aid., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Introduction: Recently, the Food and Drug Administration authorized the marketing of IQOS Tobacco Heating System as a Modified Risk Tobacco Product based on an electronic heat-not-burn technology that purports to reduce the risk., Methods: Sprague-Dawley rats were exposed in a whole-body mode to IQOS aerosol for 4 weeks. We performed the chemical characterization of IQOS mainstream and we studied the ultrastructural changes in trachea and lung parenchyma of rats exposed to IQOS stick mainstream and tissue pro-inflammatory markers. We investigated the reactive oxygen species amount along with the markers of tissue and DNA oxidative damage. Moreover, we tested the putative genotoxicity of IQOS mainstream through Ames and alkaline Comet mutagenicity assays., Results: Here, we identified irritating and carcinogenic compounds including aldehydes and polycyclic aromatic hydrocarbons in the IQOS mainstream as sign of incomplete combustion and degradation of tobacco, that lead to severe remodelling of smaller and largest rat airways. We demonstrated that IQOS mainstream induces lung enzymes that activate carcinogens, increases tissue reactive radical concentration; promotes oxidative DNA breaks and gene level DNA damage; and stimulates mitogen activated protein kinase pathway which is involved in the conventional tobacco smoke-induced cancer progression., Conclusions: Collectively, our findings reveal that IQOS causes grave lung damage and promotes factors that increase cancer risk., Implications: IQOS has been proposed as a safer alternative to conventional cigarettes, due to depressed concentration of various harmful constituents typical of traditional tobacco smoke. However, its lower health risks to consumers have yet to be determined. Our findings confirm that IQOS mainstream contains pyrolysis and thermogenic degradation by-products, the same harmful constituents of traditional cigarette smoke, and, for the first time, we show that it causes grave lung damage and promotes factors that increase cancer risk in the animal model., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In this study, a new chemiluminescent method based on the dependence of luminol light emission induced by free radicals in airborne particulate matter (PM) is proposed as a screening assay for the rapid characterization of samples from different sources based on their redox properties. This parameter is considered critical for assessing particulate matter toxicity and its impacts on human health. We propose a cell-free, luminescent assay to evaluate the redox potential of particulate matter directly on the filters employed to collect it. A joint chemometric approach based on Principal Component Analysis and Hotelling Analysis was applied to quickly sort out ambient particulate samples with a significantly different light emission profile caused by Luminol reaction. Based on Spearman correlation analysis, the association of the samples light emission intensity with their chemical composition and emission sources was attempted. The overall methodology was tested with certified reference materials and applied to two series of particulate matter samples previously subjected to thorough chemical speciation and subsequent source apportionment. The results show the effectiveness of the luminescent method, allowing the quick assessment of particulate matter oxidative potential, but providing further evidence on the complexity of the oxidative potential determination in this kind of samples. The chemometric processing of the whole dataset clearly highlights the distinct behavior among the two series of samples, the certificate standard reference materials, and the blank controls, supporting the suitability of the approach., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Safety at work should be considered as the result of the daily interaction of operators. The present research wants to analyze which factors are involved in the development of social networks about safety at work. We assumed that two relational non-technical skills, such as safety communication and safety team member support, affect the in-degree and out-degree bonds of workers in social networks. One hundred and eight workers of a warehouse were the participants of the research, in which they were asked to fill out a self-reported questionnaire. Multiple linear regression analysis was used to test the hypotheses. Results confirmed that safety communication and safety support skills play a role in determining the quantity and the quality of social bonds that workers can create at the workplace. To be specific, while safety communication was found to be associated with out-degree centrality (b = 0.24; p = 0.01), a nonsignificant relationship was found for in-degree centrality. In contrast, safety team member support was found to be associated with in-degree centrality (b = 0.28; p = 0.04). In other words, on the one hand, it was found that high levels of safety communication skills are associated with the tendency of workers to proactively search for colleagues with whom they can share information about safety. On the other hand, workers with high levels of safety support skills tend to be considered as reference points in terms of safety by colleagues, who are more prone to look for their help. Implications for both scientists and practitioners are discussed.

Effective resin disinfection is mandatory to ensure the microbiological quality of water treated by domestic softeners. The wet and sometimes warm environment inside the softener is ideal for bacteria growth. Our research was focused on the evaluation of the microbial quality of water from softeners sanitized by chlorine solutions or by electrolytic systems. We employed the heterotrophic plate count and specific tests to monitor the presence of opportunistic and pathogenic bacteria (Pseudomonas aeruginosa, Escherichia coli, enterococci, and coliforms). Completely new devices were equipped with a commercially available electrolytic system or with a newly patented one or sanitized by automatic or manual addition of chlorine solutions. In all cases, the contamination was reduced, not completely avoided. In particular, the patented electrolytic system significantly reduced bacterial proliferation in strongly contaminated devices. Our data confirm the difficulties encountered to solve the problem of microbiological quality of softened water and offer encouraging information on new possible solutions.

Duchenne muscular dystrophy (DMD) is a debilitating fatal X-linked muscle disorder. Recent findings indicate that IGFs play a central role in skeletal muscle regeneration and development. Among IGFs, insulinlike growth factor 2 (IGF2) is a key regulator of cell growth, survival, migration and differentiation. The type 2 IGF receptor (IGF2R) modulates circulating and tissue levels of IGF2 by targeting it to lysosomes for degradation. We found that IGF2R and the store-operated Ca 2+ channel CD20 share a common hydrophobic binding motif that stabilizes their association. Silencing CD20 decreased myoblast differentiation, whereas blockade of IGF2R increased proliferation and differentiation in myoblasts via the calmodulin/calcineurin/NFAT pathway. Remarkably, anti-IGF2R induced CD20 phosphorylation, leading to the activation of sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA) and removal of intracellular Ca 2+ . Interestingly, we found that IGF2R expression was increased in dystrophic skeletal muscle of human DMD patients and mdx mice. Blockade of IGF2R by neutralizing antibodies stimulated muscle regeneration, induced force recovery and normalized capillary architecture in dystrophic mdx mice representing an encouraging starting point for the development of new biological therapies for DMD., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Aim: To evaluate antithrombotic treatment patterns in patients in Italy with nonvalvular atrial fibrillation (NVAF) before and after direct oral anticoagulants (DOACs) were approved., Methods: This analysis included patients with a discharge diagnosis of NVAF in 2010 and 2014, which constituted the pre- and post-DOACs populations, respectively., Results: Approximately 90% of patients were eligible for oral anticoagulant (OAC) therapy. Overall use of OACs increased from 38% in 2010 to 45% in 2014; use of antiplatelet therapy decreased from 36 to 25%. Approximately 14% of eligible patients remained untreated., Conclusion: Although an improvement in OAC prescription was observed post-DOACs launch, treatment patterns in Italy suggest that a proportion of patients with NVAF are still undertreated or do not receive appropriate therapy.

Morphological analyses of cerebral vascularization are not only important for the characterization of the anatomical and physiological relationships between vascular and nervous tissue, but also required to understand structural modifications that occur in many pathological conditions affecting the brain. The aim of this study was to generate a three-dimensional vascular map of the cerebral hemispheres in the nude mouse brain, a widely used animal model for studying tumour biology. We used the corrosion casting (CC) technique to isolate blood vessels from 30 nude mouse brains. All casts were analysed using scanning electron microscopy (SEM), which generated quantitative data regarding vessel length and diameter as well as inter-vascular and inter-branching distances. We identified three different topographical regions: (i) the cortical region, characterized by a superficial wide sheet of vessels giving rise to terminal perforant vessels that penetrate the grey matter; (ii) the inner part of the grey matter, in which dense capillary nets form many flake-like structures extending towards the grey-white matter boundary, where perforant vessels finally change direction and form a well-defined vascular sheet; and (iii) the white matter layer, characterized by a more disorganized vascular architecture. In this study, we demonstrate the accuracy of the CC-SEM method in revealing the 3D-topographical organization of the vascular network of the normal nude mouse brain. These baseline data will serve as a reference for future anatomical investigations of pathological alterations, such as tumour infiltrations, using the nude mouse model., (© 2018 Anatomical Society.)

Purpose: Neural tube defects (NTDs) occur in 1:1000 births. The etiology is complex, with the influence of environmental and genetic factors. Environmental factors, such as folate deficiency, diabetes, or hypoxia strongly contribute to the occurrence of NTD. Also, there is a strong genetic contribution to NTD, as highlighted by the number of genes so far identified in several different developmental pathways usually altered in NTD. Each gene identified so far accounts for a small percentage of all NTD cases, indicating a very high heterogeneity., Methods: Exome sequencing was performed in seven sporadic patients with severe mielomeningocele. Novel coding variants shared by two or more patients were selected for further analysis., Results: We identified in two unrelated patients two different variants in TNIP1, a gene not previously involved in NTD whose main role is downregulation of the NF-kB pathway. One variant, c.1089T>G (p.Phe363Leu), is de novo, whereas the c.1781C>T (p.Pro594Leu) is absent in the mother, but could not be tested in the father, as he was unavailable. The latter variant is a very rare variant in the ExAC database., Conclusions: These findings suggest that TNIP1 is a new potential predisposing gene to spina bifida (SB) and its pathway needs to be investigated in human NTD in order to confirm its role and to plan appropriate counseling to families.

Chronic subdural hematomas mainly occur amongst elderly people and usually develop after minor head injuries. In younger patients, subdural collections may be related to hypertension, coagulopathies, vascular abnormalities, and substance abuse. Different techniques can be used for the surgical treatment of symptomatic chronic subdural hematomas : single or double burr-hole evacuation, with or without subdural drainage, twist-drill craniostomies and classical craniotomies. Failure of the brain to re-expand, pneumocephalus, incomplete evacuation, and recurrence of the fluid collection are common complications following these procedures. Acute subdural hematomas may also occur. Rarely reported hemorrhagic complications include subarachnoid, intracerebral, intraventricular, and remote cerebellar hemorrhages. The causes of such uncommon complications are difficult to explain and remain poorly understood. Overdrainage and intracranial hypotension, rapid brain decompression and shift of the intracranial contents, cerebrospinal fluid loss, vascular dysregulation and impairment of venous outflow are the main mechanisms discussed in the literature. In this article we report three cases of different post-operative intracranial bleeding and review the related literature.

Brain microvascular endothelial cells (BMVECs) have an important role in the constitution of the blood-brain barrier (BBB). The BBB is involved in the disease processes of a number of neurological disorders in which its permeability increases. Isolation of BMVECs could elucidate the mechanism involved in these processes. This protocol describes how to isolate and expand human and mouse BMVECs. The procedure covers brain-tissue dissociation, digestion and cell selection. Cells are selected on the basis of time-responsive differential adhesiveness to a collagen type I-precoated surface. The protocol also describes immunophenotypic characterization, cord formation and functional assays to confirm that these cells in endothelial proliferation medium (EndoPM) have an endothelial origin. The entire technique requires ∼7 h of active time. Endothelial cell clusters are readily visible after 48 h, and expansion of BMVECs occurs over the course of ∼60 d.

Background: Brain microvascular endothelial cells (BMVECs) constitute the primary limitation for passage of ions and molecules from the blood into the brain through the blood brain barrier. Numerous multi-step procedures for isolating and culturing BMVECs have been described. However, each one demonstrates major limitations in purity of culture and/or low proliferation rate. Our goal was to study the efficiency of our pending patent medium, Endothelial Proliferation Medium (EndoPM), on the isolation and purification of human and murine BMVECs., Methods: BMVECs, cultured in EndoPM were compared to those cultured in a commercial medium EBM. Cultures were characterized by flow cytometric analysis, lineage differentiation, the ability to form tube-like structure, immunofluorescence, molecular analyses and also in an in vivo model assay. Moreover permeability was assayed by monitoring the passage of Dextran-FITC through a tight monolayer of BMVECs grown to confluence in Boyden chambers. One way Anova two-tailed test was utilized for all statistical analyses., Results: The properties of ECs in human and murine BMVECs is confirmed by the expression of endothelial markers (CD31, CD105, CD146, Tie-2 and vWF), of representative proangiogenic genes (ICAM1, VCAM1 and integrin ITGAV), of considerable tube-forming ability, with low-density lipoprotein uptake, eNOS and GLUT-1 expression. Furthermore cells are able to express markers of the junctional architecture as VE-cadherin, β-catenin and Claudin-5 and greatly reduce dextran permeability as barrier functional test. Moreover BMVECs spontaneously organize in vascular-like structures and maintain the expression of endothelial markers in an in vivo xenograft model assay. The significant effect of EndoPM is confirmed by the study of proliferation index, survival index and the behaviour of BMVECs and fibroblasts in co-culture conditions., Conclusion: Herein we describe a simple and reproducible method for the isolation and expansion of human and mouse BMVECs, based on a newly formulated medium (EndoPM) with optimized concentration of growth factors (EGF, FGF-2 and Bovine Brain Extract-BBE). This procedure should facilitate the isolation and expansion of human and mouse BMVECs with extended lifetime, good viability and purity. This approach may provide an effective strategy to aid phenotypical and functional studies of brain vessels under physiological and pathological conditions.

Object: Human dura mater is the most external meningeal sheet surrounding the CNS. It provides an efficient protection to intracranial structures and represents the most important site for CSF turnover. Its intrinsic architecture is made up of fibrous tissue including collagenic and elastic fibers that guarantee the maintenance of its biophysical features. The recent technical advances in the repair of dural defects have allowed for the creation of many synthetic and biological grafts. However, no detailed studies on the 3D microscopic disposition of collagenic fibers in dura mater are available. The authors report on the collagenic 3D architecture of normal dura mater highlighting the orientation, disposition in 3 dimensions, and shape of the collagen fibers with respect to the observed layer., Methods: Thirty-two dura mater specimens were collected during cranial decompressive surgical procedures, fixed in 2.5% Karnovsky solution, and digested in 1 N NaOH solution. After a routine procedure, the specimens were observed using a scanning electron microscope., Results: The authors distinguished the following 5 layers in the fibrous dura mater of varying thicknesses, orientation, and structures: bone surface, external median, vascular, internal median, and arachnoid layers., Conclusions: The description of the ultrastructural 3D organization of the different layers of dura mater will give us more information for the creation of synthetic grafts that are as similar as possible to normal dura mater. This description will be also related to the study of the neoplastic invasion.

In this morphological study, we report on the three-dimensional microvascular architecture constituting the toes of a patient affected by diabetic microangiopathy. We applied corrosion casting (CC) technique to the toes of a patient affected by Type 2 diabetes, who underwent surgery for explantation of inferior left limb due to necrotic processes of soft tissues. The toes of a foot traumatically explanted in a motorcycle accident were kept as controls. According to technical protocols, toes were injected with a low-viscosity acrylic resin (Mercox) through the major digital artery, tissues were corroded in KOH solution (8%), and resulting casts processed for SEM observations. Already at low magnification, in diabetic toes, we found an impairment of the linear track-like disposition of the vessels of plantar side, with signs of vascular disruption and obliterations, stopped resin, and leakages. Capillaries under the nail and a lot of vascular villi in eponychium and nail borders were damaged, and vascular regression phenomena acting on them were clearly visible. Resin leakages and impairment of normal vascular architecture were also observed in the root of the nail. This preliminary report represents only the first step for further investigations regarding morphological three-dimensional appearance of diabetic microangiopathy. CC and scanning electron microscopy technique well documented these morphological modifications, highlighting on both structural and ultrastructural features of diabetic toes microvessels. In conclusion, our qualitative data try to better focus on the pathophysiological mechanisms involved in diabetic dermopathy and microangiopathy, proposing CC as useful method to investigate on them., (Copyright © 2010 Wiley-Liss, Inc.)

Methyl-CpG-binding protein 2 (MECP2) gene mutations have been identified in girls with Rett syndrome and in boys with heterogeneous neuropsychiatric disorders. Because of the limited or inconsistent data reported in literature, the role of methyl-CpG-binding protein 2 gene in the pathogenesis of mental retardation and pervasive developmental disorders needs further study. We scanned methyl-CpG-binding protein 2 gene in 99 Italian patients with pervasive developmental disorder or with nonsyndromal mental retardation. Four methyl-CpG-binding protein 2 gene mutations were found: 2 in 4 girls with Rett disorder, the others in 2 girls with mental retardation. The wide phenotypic spectrum and the variants of methyl-CpG-binding protein 2 gene, which may play an important role in gene regulation and neurodevelopment, justify the literature's interest particularly in girls.

Since the dorsal root ganglia represent the first structure of pain modulation, they are the target of the newest therapies of neuropathic pain. Between these, pulsed radiofrequency (PRF) has been described among the promising non-invasive methods. Although the results encourage the clinical use of this procedure, their mechanism of action is still unclear. Aim of our study was to analyze acute effects of PRF on the rat lumbar ganglion and on nervous fibres running inside it. Clinical works describe PRF treatment as a technique without any visible neurological deficit. The few disposable histological works are contractictory: some describe no signs of cellular damage and some demonstrate visible intracellular modifications. A total of 20 male Wistar rats were deeply anesthesized. Ten were positioned in a stereotactic system, and exposed to PRF at 2 Hz for 30 s after exposition of paravertebral muscles and positioning of a stimulation needle on left L4 ganglion. The other ten were used as controls. After 1 h, the left dorsal root ganglions L3, L4, L5 of the 20 animals were explanted, fixed in 2.5% Karnowsky solution and prepared for light and transmission electron microscopy. At light microscopy no differences between treated and control animals were observed; at transmission electron microscopy, instead, it was possible to observe that T gangliar cells contained an abnormal abundant smooth reticulum with enlarged cisternae and numerous vacuoles; myelinated axons presented pathological features and their myelin coverage was not adherent. Instead, unmyelinated axons appeared normal in shape and dimension and the Schwann cells surrounding it had intact plasmamembrane. Our results, obtained at acute stage, reveal that the PRF procedure should destroy the myelin envelope of nervous fibres. Further future studies, at chronic stage, should give other information on the prognosis of the myelinic damage.

Background: The antimicrobial activity of intrathecal baclofen was investigated. Several different microorganisms were used: Staphylococcus aureus (beta-lactamase-positive and beta-lactamase-negative strains); S epidermidis; Enterococcus faecalis; Klebsiella pneumoniae; Escherichia coli; Pseudomonas aeruginosa; and Candida albicans., Methods: Three experimental approaches were used to assess baclofen antimicrobial activity: (1) determination of the MIC; (2) determination of the MBC; and (3) kinetic time-kill assay. Experiments were performed according to current methods of the NCCLS., Results: As compared with control organisms exposed to physiologic saline, organisms exposed to baclofen over a 10-day period failed to reduce the number of viable cells by at least 3 log(10), as requested by NCCLS criteria., Conclusions: Because the viability of the investigated organisms was not reduced over that of microbial suspensions exposed to physiologic saline, we conclude that intrathecal baclofen has no measurable activity against different bacterial species and C albicans.

Ocular involvement is a prevalent feature in mitochondrial diseases. Leber's hereditary optic neuropathy (LHON) and dominant optic atrophy (DOA) are both non-syndromic optic neuropathies with a mitochondrial etiology. LHON is associated with point mutations in the mitochondrial DNA (mtDNA), which affect subunit genes of complex I. The majority of DOA patients harbor mutations in the nuclear-encoded protein OPA1, which is targeted to mitochondria and participates to cristae organization and mitochondrial network dynamics. In both disorders the retinal ganglion cells (RGCs) are specific cellular targets of the degenerative process. We here review the clinical features and the genetic bases, and delineate the possible common pathomechanism for both these disorders.

Oculomotor dysfunctions are hidden causes of invalidity following whiplash injury. Many patients with whiplash injury grade II present oculomotor dysfunctions related to input disturbances of cervical or vestibular afferents. We used static posturography to investigate 40 consecutive patients with whiplash injury grade II and oculomotor dysfunctions. We demonstrated a relation between length and surface of body sway: the surface value (A) was higher than the length value (L) and this led to an open graph of body sway in the statokinesigram. Oculomotor rehabilitation can resolve the impairment of vestibular function but if therapy is delayed or the patient has been wearing an orthopaedic neck collar, more therapeutic sessions are required. In conclusion, without rehabilitation of the oculomotor muscles other therapies are not sufficient to recover the impairment caused by whiplash injury.

Up to now, baclofen (a GABA(B) receptor agonist) has been used for the treatment of severe spasticity unresponsive to oral antispasmodics. Although in humans it is usually administered at 2 mg/ml, the dosage to be used in the treatment of other diseases is unknown. For this reason, it is important to determine the safe maximum dosage and toxicity at the clinically used concentration. Primary cortical neurons represent a useful model to test the safety of baclofen. We performed a colorimetric assay (MTT test) as well as electron microscopy investigations, to determine neuronal survival after the treatment with baclofen at a concentration of 2 and 4 mg/ml. Our results demonstrated that, in our experimental model, neither concentration affected neuronal survival. Considering the above results, we can conclude that at the used concentrations, this drug is safe and its clinical use should be encouraged.

Corrosion casting combined with scanning electron microscopy (SEM) has been widely used to study the morphofunctional aspects of microcirculation in many organs. In this study, we present an optimization of the corrosion casting (CC) technique associating it with NaOH 1 N maceration method to obtain a clear visualization of the relationships existing between the microvascular architecture of an organ and its extracellular matrix. Briefly, experiments were performed macerating the tissue previously injected with a low viscosity acrylic resin in 1 N NaOH and then observing it at SEM. In this study, we present an application of this technique to better evaluate the extracellular components of the vascular wall in medium-sized and capillary vessels both in skin and in kidney. The results obtained yielded clear images of the three-dimensional layout of medium-sized and capillary vessels in comparison with the extracellular environment. Furthermore, detailed information was obtained on the three-dimensional layout of fibers constituting the walls of venules, arterioles, and capillaries. In addition, the tubular collagenic structures surrounding the excretory tubules of the kidney and the dermal glands of the skin were depicted and their relationships with their vascular supply described in detail., ((c) 2006 Wiley-Liss, Inc.)

It has been proposed that European mitochondrial DNA (mtDNA) haplogroups J and K, and their shared 10398G single-nucleotide polymorphism (SNP) in the ND3 gene, are protective from Parkinson's disease (PD). We evaluated the distribution of the different mtDNA haplogroups in a large cohort of 620 Italian patients with adult-onset (>50, <65 years of age) idiopathic PD vs two groups of ethnic-matched controls. Neither the frequencies of haplogroup J nor that of 10398G were significantly different. However, the frequency of haplogroup K was significantly lower in PD. Stratification by sex and age indicated that the difference in the distribution of haplogroup K was more prominent in >50 year old males. In spite of the common 10398G SNP, haplogroups J and K belong to widely diverging mitochondrial clades, a consideration that may explain the different results obtained for the two haplogroups in our cohorts. Our study suggests that haplogroup K might confer a lower risk for PD in Italians, corroborating the idea that the mitochondrial oxidative phosphorylation pathway is involved in the susceptibility to idiopathic PD.

We performed a morphological scanning electron microscope (SEM) study to describe the fine structure and disposition of collagenous tissue in the human toe. After therapeutic amputation of a human right leg, we applied the Othani maceration technique to the skin of three toes surgically explanted from the foot. We distinguished eight cutaneous regions and focused on some specialized collagenous structures differing in the thickness of the skin. The eight areas investigated were: the dorsal skin, the eponychium, the perionychium, the hyponychium, the region under the visible nail, the nail root, the plantar skin and finally the toe tip. Each of these areas is characterized by a distinctive collagenous surface disposition, with some peculiar features mostly related to dermal papillae. At high magnification, we observed the spatial arrangement of the collagen fibers constituting the top of the dermal papillae that represents the attachment site of the proliferative basal layer of the epidermis. We also noted an impressive density of collagen fibers throughout the thickness of the dermal layer, organized in specialized structures and constituting the skeleton of dermal thermoreceptorial corpuscles or sweat glands. A combination of SEM and Ohtani technique disclosed the three-dimensional architecture of the collagenous matrix of tarsal skin under physiologic conditions, giving a detailed description of the most reactive tissue during pathologic processes.

Scanning electron microscopic observation of corrosion casts is the finest technique to describe spatial patterns of microvessels in many organs, giving a readily interpreted representation of their vascular architecture without interference from surrounding tissues. We focused on the renal cortex of guinea pigs to make an in-depth morphological analysis of structural and ultrastructural details left by the cells on the resin cast. In addition, we made a qualitative description of normal variants usually observed in glomerular disposition, arteriolar morphology or capillary arrangement in the space to shed more light on the relationship between vascular tissue and surrounding cells. The study also disclosed some examples of vascular adaption to physiological and pathological conditions occurring in renal microvessels such as many systems essential to flow regulation, filtration and excretory processes. At lower magnification, all major vessels can be readily distinguished: interlobar, arciform and interlobular arteries and veins, along with a web of peritubular and capsular capillaries. At higher magnification, the glomeruli become visible and the afferent and efferent arteries and the tortuosity the inner vessels can be distinguished. In some of them, the resin, due to the narrowing sizes, suddenly stopped leaving a half-casted glomerulus. This helped to reveal its internal circulation characterized by thin capillaries with a high degree of bi or trifurcation. In addition, we confirmed the close correspondence between cellular ultrastructural detail (pores, corrugations of cellular membrane, perivascular cell branches) and the impressions left on the resin visible only at high magnifications.

We describe the case of a 10-year-old girl with two epileptic seizures and subcontinuous spike-waves during sleep, who presented unusual side-effects related to clobazam (CLB) monotherapy. High plasma levels of N-desmethyl-clobazam (N-CLB), the major metabolite of CLB were detected. The patient and her parents underwent molecular analysis of the CYP2C19 gene, which may be implicated in the metabolism of this drug. Our patient presents one copy of the most common mutation (CYP2C19*2) affecting the activity of the isoenzyme and probably another rare or private mutation. CLB and N-CLB plasma level dosages and molecular analysis may be useful when a poor metabolic condition is suspected.

Vascular and bronchial endocasts represent a useful instrument to study the ramification pattern of these structures. Casts have been made from different materials, such as waxes in ancient times and, more recently, silicon-like compounds or resins (see e.g. Mercox) to study the finest details. These techniques are valuable for small specimens, whereas they are inadequate for very large organs, where technical difficulties require the development of specific instrumentation. In this study we present a new simple injection technique, based on expanded polyurethane, which allows preparing vascular and bronchial trees for macroscopic and microscopic studies. The new injection technique is very easy to carry out, since the propulsion is provided by compressed air, and it does not require special instrumentation. To this aim, endocasts of the entire tracheal-bronchial tree and casts of vascular kidney from different animals were prepared. The specimens have a very low weight, show the finest ramifications, and are very stable and resistant to mechanical stress. To examine microscopically the details of the casts, specimens from the kidney cast were also analyzed by scanning electron microscopy, revealing good preservation of microcirculatory structures, functional sphincters and endothelial cell impressions. Therefore, the technique may be useful for macroscopic studies of large specimens, retaining sufficiently fine details.