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OBJECTIVE: Head and neck region is involved in a high percentage of malignant lesions, and oral squamous cell carcinoma (OSCC) is undoubtedly the most frequently found, accounting for over 90% of malignant tumors. Hormone receptor overexpression, like Estrogen Receptor (ER), Progesterone Receptor (PR) and Endothelial Growth Factor Receptor (EGFR), and signaling have been related to the pathogenesis of OSCC. For metastasis of OSCC, Cancer Stem Cells (CSCs) undergo epithelial to mesenchymal transition (EMT) under the influence of growth factors, cytokines, and regulation of cadherins from the tumor’s microenvironment. In this context, the stem cells may become a potential therapeutic target for OSCC through modulation of cytokines and RAS pathway, which is involved in intracell signal transduction. The objective of this study was to suggest an experimental steroidogenic model for OSCC in translational research. PATIENTS AND METHODS: Dental-derived Stem Cells (D-dSCs) have been obtained from apical papilla tissue that surrounds the developing tooth of healthy donors and cultured in vitro. The cells have been exposed to different concentrations of Estradiol (E2 - 10 nM and 40 nM) in order to verify their response. The number of cells and cell viability has been evaluated up to 96 hours of treatment. RESULTS: The results showed that cell growth was increased under estradiol treatments compared with cells maintained without estradiol. Moreover, no significant difference in cell death levels was detected among treatments. CONCLUSIONS: This work underlines as D-dSCs could represent a useful steroidogenic model for the development of the target and gene therapies in OSCC.

Concentrated Growth Factors (CGF) is a platelet rich preparation that has the important feature of a tight fibrin network and containing a large number of growth factors possessing great regenerative potentialities [1]. The regeneration of nervous system is one of the mail goal of regenerative medicine. The aim of this study is to test the in vitro CGF effects on both differentiated and undifferentiated SH-SY5Y cells, derived from human neuroblastoma. To induce differentiation, SH-SY5Y cells have been treated with Retinoic Acid (RA) 10µM, in both basal and complete medium and in the presence and absence of CGF. After 72 hours, different parameters have been investigated: the morphological characteristics of the cells, the cell proliferation, the cellular vitality using the MTT test, the CGF and/or RA differentiation property and the immunocytochemical analysis of neuronal specific markers (NeuN, Sinaptophisine, β-III-tubulin, Nestin). Moreover the NGF (Nerve Growth Factor) and BDNF (Brain Derived Growth Factor) release have been assayed by ELISA test. Our results obtained suggest that treatment with CGF, also used alone, positively affects cell differentiation and neuronal phenotype regulating the expression of the neuronal markers and improving the outgrowth of neurites. Taken together these results seems to be promised into new approaches for neuronal regeneration using platelet preparations., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

Chemokines are involved in neuroinflammation and contribute to chronic pain processing. The new chemokine prokineticin 2 (PROK2) and its receptors (PKR1 and PKR2 ) have a role in inflammatory pain and immunomodulation. In the present study, we investigated the involvement of PROK2 and its receptors in neuropathic pain.Effects of single, intrathecal, perineural and s.c. injections of the PKR antagonist PC1, or of 1 week s.c. treatment, on thermal hyperalgesia and tactile allodynia was evaluated in mice with chronic constriction of the sciatic nerve (CCI). Expression and localization of PROK2 and of its receptors at peripheral and central level was evaluated 10 days after CCI, following treatment for 1 week with saline or PC1. IL-1β and IL-10 levels, along with glia activation, were evaluated.Subcutaneous, intrathecal and perineural PC1 acutely abolished the CCI-induced hyperalgesia and allodynia. At 10 days after CCI, PROK2 and its receptor PKR2 were up-regulated in nociceptors, in Schwann cells and in activated astrocytes of the spinal cord. Therapeutic treatment with PC1 (s.c., 1 week) alleviated established thermal hyperalgesia and allodynia, reduced the injury-induced overexpression of PROK2, significantly blunted nerve injury-induced microgliosis and astrocyte activation in the spinal cord and restored the physiological levels of proinflammatory and anti-inflammatory cytokines in periphery and in spinal cord.The prokineticin system contributes to pain modulation via neuron-glia interaction. Sustained inhibition of the prokineticin system, at peripheral or central levels, blocked both pain symptoms and some events underlying disease progression.

The pineal gland is a small neuroendocrine organ involved primarily in the circadian rhythm by the secretion of melatonin [1]. In addition, the pain modulatory properties of melatonin are generally recognized but its involvement in neuropathic pain regulation is not fully understood. In fact, it is known that the activation of the endogenous melatonin system in the spinal cord can reduce the generation, development and maintenance of central sensitization [2]. Moreover, melatonin showed an analgesic effect, in fact several works in animals [2] and in humans [3] underline its ability to inhibit hyperalgesia. In particular, intracerebroventricular and intraperitoneal melatonin, with its higher doses, produces a blockade of thermal hyperalgesia in mice with partial tight ligation of the sciatic nerve. The aim of our work is to characterize the morphological changes in peripheral structures, such as plantar skin and dorsal root ganglia (DRG) of rats in a neuropathic pain model (chronic constriction injury) after a single melatonin treatment monitoring the behaviour and the changes in NO-system using immunohistochemical techniques. The behavioural results show an increase of withdrawal latency during plantar test already after 30 min from melatonin administration. The immunohistochemical results suggest that melatonin plays a crucial role in keratinocytes-mediated neuropathic pain transmission through the modulation of nitroxidergic system, which could have also a protective role at this site. In addition, at DRG level the NO-system is maintained at low level. These results suggest that melatonin administration or modulation of pineal gland activity may have clinical utility in neuropathic pain therapy in the future., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

Calcitonin gene-related peptide (CGRP) is a vasodilatory peptide that has been detected at high levels in the skin, blood, and cerebrospinal fluid (CSF) under a variety of inflammatory and chronic pain conditions, presumably derived from peptidergic C and Aδ innervation. Herein, CGRP immunolabeling (IL) was detected in epidermal keratinocytes at levels that were especially high and widespread in the skin of humans from locations afflicted with postherpetic neuralgia (PHN) and complex region pain syndrome type 1 (CRPS), of monkeys infected with simian immunodeficiency virus, and of rats subjected to L5/L6 spinal nerve ligation, sciatic nerve chronic constriction, and subcutaneous injection of complete Freund's adjuvant. Increased CGRP-IL was also detected in epidermal keratinocytes of transgenic mice with keratin-14 promoter driven overexpression of noggin, an antagonist to BMP-4 signaling. Transcriptome microarray, quantitative Polymerase Chain Reaction (qPCR), and Western blot analyses using laser-captured mouse epidermis from transgenics, monolayer cultures of human and mouse keratinocytes, and multilayer human keratinocyte organotypic cultures, revealed that keratinocytes express predominantly the beta isoform of CGRP. Cutaneous peptidergic innervation has been shown to express predominantly the alpha isoform of CGRP. Keratinocytes also express the cognate CGRP receptor components, Calcitonin receptor-like receptor (CRLR), Receptor activity-modifying protein 1 (RAMP1), CGRP-receptor component protein (RCP) consistent with known observations that CGRP promotes several functional changes in keratinocytes, including proliferation and cytokine production. Our results indicate that keratinocyte-derived CGRPβ may modulate epidermal homeostasis through autocrine/paracrine signaling and may contribute to chronic pain under pathological conditions.

Mercuric chloride (HgCl2) induces acute renal failure associated to tubular impairment in experimental animals and humans. Stress proteins are a superfamily of proteins, comprising heat- shock proteins (HSP) and glucose-regulated proteins (GRP), enhanced or induced in the kidney in response to stress. They act as molecular chaperones that protect organelles and repair essential proteins which have been denatured during adverse conditions. The involvement of stress proteins in mercury-nephrotoxicity has not yet been well clarified. This study was undertaken to detect the tubular distribution of four stress proteins (HSP25, HSP60, GRP75, HSP72) in the rat kidney injected with HgCl2 and to quantify lysosomal and mitochondrial changes in straight proximal tubules, the main mercury target. Sprague-Dawley rats were administered i.p. with progressive sublethal doses of HgCl2 (0.25 mg/kg, 0.5 mg/kg, 1 mg/kg and 3.5 mg/kg) or saline (as controls) and sacrificed after 24 h. In dosages over 0.50 mg/kg, stress proteins increased and changed localization in a dose-dependent manner. HSP25 was focally expressed in altered proximal tubules at 1 mg/kg but in the macula densa it was at 3.5 mg/kg. HSP60 and GRP75 were intense in the nucleus and cytoplasm of proximal tubules but moderate in distal tubules. HSP72 was induced in distal tubules after low exposures but in proximal tubules it happened at the highest dose. Moreover, a significant increase in lysosomal and total mitochondria (normal and with broken cristae) area and density were progressively found after HgCl2 treatments. Stress proteins could represent sensitive biomarkers that strongly correlate with the degree of oxidative injury induced by HgCl2 in the rat proximal tubules.

Physiology of orofacial pain pathways embraces primary afferent neurons, pathologic changes in the trigeminal ganglion, brainstem nociceptive neurons, and higher brain function regulating orofacial nociception. The goal of this study was to investigate the nitroxidergic system alteration at brainstem level (spinal trigeminal nucleus), and the role of peripheral P2 purinergic receptors in an experimental mouse model of pediatric inflammatory orofacial pain, to increase knowledge and supply information concerning orofacial pain in children and adolescents, like pediatric dentists and pathologists, as well as oro-maxillo-facial surgeons, may be asked to participate in the treatment of these patients. The experimental animals were treated subcutaneously in the perioral region with pyridoxalphosphate-6-azophenyl-2 ′ ,4 ′ -disulphonic acid (PPADS), a P2 receptor antagonist, 30 minutes before formalin injection. The pain-related behavior and the nitroxidergic system alterations in the spinal trigeminal nucleus using immunohistochemistry and western blotting analysis have been evaluated. The local administration of PPADS decreased the face-rubbing activity and the expression of both neuronal and inducible nitric oxide (NO) synthase isoforms in the spinal trigeminal nucleus. These results underline a relationship between orofacial inflammatory pain and nitroxidergic system in the spinal trigeminal nucleus and suggest a role of peripheral P2 receptors in trigeminal pain transmission influencing NO production at central level. In this way, orofacial pain physiology should be elucidated and applied to clinical practice in the future.

Bone marrow and adipose tissue human mesenchymal stem cells were seeded in highly performing 3D gelatin–chitosan hybrid hydrogels of varying chitosan content in the presence of human platelet lysate and evaluated for their proliferation and osteogenic differentiation. Both bone marrow and adipose tissue human mesenchymal stem cells in gelatin–chitosan hybrid hydrogel 1 (chitosan content 8.1%) or gelatin–chitosan hybrid hydrogel 2 (chitosan 14.9%) showed high levels of viability (80%–90%), and their proliferation and osteogenic differentiation was significantly higher with human platelet lysate compared to fetal bovine serum, particularly in gelatin–chitosan hybrid hydrogel 1. Mineralization was detected early, after 21 days of culture, when human platelet lysate was used in the presence of osteogenic stimuli. Proteomic characterization of human platelet lysate highlighted 59 proteins mainly involved in functions related to cell adhesion, cellular repairing mechanisms, and regulation of cell differentiation. In conclusion, the combination of our gelatin–chitosan hybrid hydrogels with hPL represents a promising strategy for bone regenerative medicine using human mesenchymal stem cells.

A nonphysiological repair of the lesioned nerve leading to the formation of neurinomas, altered nerve conduction, and spontaneous firing is considered the main cause of the events underlying neuropathic pain. It was investigated whether neural stem cell (NSCs) administration could lead to a physiological nerve repair, thus to a reduction of neuropathic pain symptoms such as hyperalgesia and allodynia in a well-established model of this pain (sciatic nerve chronic constriction injury [CCI]). Moreover, since we and others showed that the peripheral nerve lesion starts a cascade of neuroinflammation-related events that may maintain and worsen the original lesion, the effect of NSCs on sciatic nerve pro- and antiinflammatory cytokines in CCI mice was investigated. NSCs injected intravenously, when the pathology was already established, induced a significant reduction in allodynia and hyperalgesia already 3 days after administration, demonstrating a therapeutic effect that lasted for at least 28 days. Responses changed with the number of administered NSCs, and the effect on hyperalgesia could be boosted by a new NSC administration. Treatment significantly decreased proinflammatory, activated antiinflammatory cytokines in the sciatic nerve, and reduced spinal cord Fos expression in laminae I-VI. Moreover, in NSC-treated animals, a reparative process and an improvement of nerve morphology is present at a later time. Since NSC effect on pain symptoms preceded nerve repair and was maintained after cells had disappeared from the lesion site, we suggest that regenerative, behavioral, and immune NSC effects are largely due to microenvironmental changes they might induce at the lesion site. © 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

OBJECTIVE: Structural alterations in the microcirculation may be considered an important mechanism of organ damage. An increased media-to-lumen ratio of subcutaneous small resistance arteries has been demonstrated to predict the development of cardiocerebrovascular events in hypertensive patients. Alterations in the structure of small cerebral arteries have been demonstrated in animal models of experimental or genetic hypertension. However, no evaluation with reliable techniques has ever been performed in humans. DESIGN AND METHODS: Twenty-eight participants were included in the present study: they were 13 hypertensive patients and 15 normotensive individuals. All participants underwent a neurosurgical intervention for benign or malign tumors. A small portion of morphologically normal cerebral tissue was excised from surgical samples and examined. Cerebral small resistance arteries (relaxed diameter around 200 mum) were dissected and mounted on an isometric and isobaric myograph, and the tunica media to internal lumen ratio was measured. In addition, cerebral cortical microvessel density (MVD) was also evaluated. The tissue was sectioned and stained for CD31, and MVD was measured with an automated image analyzer (percentage of area stained). Blood pressure values were evaluated, before surgical intervention, by standard sphygmomanometry. RESULTS: M/L was significantly greater and MVD significantly lower in hypertensive patients than that in normotensive individuals. No difference between groups in collagen content or mechanical properties of cerebral small arteries was observed. CONCLUSION: Our results indicate that structural alterations of small cerebral vessels are present in hypertensive patients compared with normotensive individuals, similar to those previously observed in subcutaneous small arteries.

Neuropathic pain consequent to peripheral injury is associated with local inflammation and overexpression of nitric oxide synthases (NOS) and inflammatory cytokines in locally recruited macrophages, Schwann and glial cells. We investigated the time course and localization of nitric oxide synthases (NOS) and cytokines in the central (spinal cord and thalamus) and peripheral nervous system (nerve and dorsal root ganglia), in a mouse model of mononeuropathy induced by sciatic nerve chronic constriction injury. ATP is recognized as an endogenous pain mediator. Therefore we also evaluated the role of purinergic signalling in pain hypersensitivity employing the P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), on pain behaviour, NOS and cytokines. The PPADS daily administration starting on day 3 after injury dose- and time-dependently decreased both tactile allodynia and thermal hyperalgesia. PPADS (25mg/kg) completely reversed nociceptive hypersensitivity and simultaneously reduced the increased NO/NOS system and IL-1beta in both peripheral (injured sciatic nerve and L4-L6 ipsilateral dorsal root ganglia) and central steps of nervous system (L4-L6 spinal cord and thalamus) involved in pain signalling. IL-6 was overexpressed only in the peripheral nervous system and PPADS prolonged administration reduced it in sciatic nerve. In conclusion, we hypothesize that NO/NOS and IL-1beta have a pronociceptive role in this neuropathy model, and that purinergic antagonism reduces pain hypersensitivity by inhibiting their overactivity.

The reaction between an un-functionalized conjugated diene and a nitroarene under CO pressure, catalyzed by Ru-3(CO)(12)/Ar-BIAN (Ar-BUN = bis(arylimino)acenaphthene), affords the corresponding allylic amine (1), the hetero-Diels-Alder adduct (oxazine) (2), and the N-arylpyrrole (3) in different ratios depending on the experimental conditions. The synthesis of the allylic amine involves an intermolecular catalytic C-H functionalization by a transition metal complex. Compounds I and 2 are primary products of the reaction, whereas 3 derives from a following reaction of 2. By running the reaction at 120 degreesC, the decomposition of 2 to 3 is completely suppressed, allowing for the isolation of 2 in good yields. On the contrary, at 200 degreesC 2 is completely transformed into 3 during the reaction. The selectivity in allylic amine is somewhat sensitive to the experimental conditions and always ranges between 15 and 25%. Electron-withdrawing substituents on the nitroarene give better results, but electron-donating ones slow the reaction and give lower selectivities. Steric hindrance on the nitroarene strongly retards the reaction, but use of 2-methylnitrobenzene allowed for the isolation and X-ray structural characterization of a resting state of the catalytic system, Ru[N(o-CH3C6H4)C(O)N(o-CH3C6H4)C(O)](CO)(2)(Ph-BIAN) (9)