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The coastal lagoon of Lesina, is situated on the Italian southern Adriatic coast, in a primarily rural area. Agriculture is the primary economic activity, while the lagoon supports only small scale fisheries and one aquafarm enterprise. However, the lagoon ecological state shows a slow but constant deterioration. In this study, we a) assess the causes for this decline; b) evaluate why the existing regulations have failed to protect the environment from this deterioration; c) explore the potential for community involvement in the management of the lagoon; d) formulate suggestions for developing a management plan that will ensure the preservation of the lagoon and the well-being of the community, according to a shared stakeholders’ vision. For this purpose, available social, economic and physical data were gathered, organized and successively used to identify the causes for the lagoon deterioration. We found that the main causes are: increased discharge of sediments and nutrients from inland activities; unmanaged fishing; loss of human capital; unclear regulations due to vertical and horizontal lack of coordination among authorities. Further, three workshops were conducted with local stakeholders to identify the multiple problems affecting the lagoon area and discuss possible shared solutions. The workshops highlighted that participants perceived the lagoon as an unexploited source of income, mostly in terms of nature based tourism, and suggested that negative externalities caused by the inland activities and mismanagement of the fishery sector should be accounted for by the authorities. However, the mapping of stakeholders indicates that the tourist sector in the area is not developed enough to effectively promote in a bottom-up fashion the development of a management plan of the lagoon area, which could encourage sustainable tourism by protecting the lagoon and generating economic development for the area.

The transplantation of pancreatic islets is an innovative and intriguing therapeutic option for the long term treatment of type-1 diabetes (Remuzzi et al., 2009). Unfortunately, their clinical feasibility is limited by the great number of islets necessary to achieve glycaemic control and their short survival. A possible means to improve the performance of this technique can be represented by Mesenchymal Stem Cells (MSCs), adult stem cells alrady known to support the survival of different cellular populations (Scuteri et al., 2014). In this work the ability of Mesenchymal Stem Cells (MSCs) to improve the feasibility of this approach was verified into an in vivo model represented by Streptozotocin-induced diabetic rats. We compared 5 different groups (8 rats/group): a) healthy controls; b) Diabetic rats; c) Diabetic rats transplanted with pancreatic islets (3000); d) Diabetic rats cotransplanted with pancreatic islets (2000) and MSCs (106); Diabetic rats treated with MSCs (106). Transplantations were performed after the assessment of neuropathic signs, represented by a decreased Nerve Conduction Velocity (NCV) and an impairment of nociceptive thermal and mechanical thresholds. The same parameters were evaluated two months after the transplantation. Diabetic rats transplanted only with pancreatic islets, or co-transplanted with MSCs and a suboptimal number of pancreatic islets, showed a significant glycaemia value reduction, an improvement of thermal and mechanical sensitivity, and an improvement of NCV with respect to diabetic-untreated rats. No differences were observed between diabetic rats and diabetic rats treated with only MSCs. In conclusion, we demonstrated that co-transplantation with MSCs reduces the number of pancreatic islets needed to reach glycaemic control, and induces the regression of painful neuropathy signs, thus ameliorating diabetes complications management. Granted by MIUR – FIRB Futuro in Ricerca 2008 Prot. N° RBFR08VSVI_001., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

We studied the DNA fragmentation induced in human fibroblasts by iron-ion beams of two different energies: 115 MeV/nucleon and 414 MeV/nucleon. Experimental data were obtained in the fragment size range 1-5700 kbp; Monte Carlo simulations were performed with the PARTRAC code; data analysis was also performed through the Generalized Broken Stick (GBS) model. The comparison between experimental and simulated data for the number of fragments produced in two different size ranges, 1-23 kbp and 23-5700 kbp, gives a satisfactory agreement for both radiation qualities. The Monte Carlo simulations also allow the counting of fragments outside the experimental range: The number of fragments smaller than 1 kbp is large for both beams, although with a strong difference between the two cases. As a consequence, we can compute different RBEs depending on the size range considered for the fragment counting. The PARTRAC evaluation takes into account fragments of all sizes, while the evaluation from the experimental data considers only the fragments in the range of 1-5700 kbp. When the PARTRAC evaluation is restricted to this range, the agreement between experimental and computed RBE values is again good. When fragments smaller than 1 kbp are also considered, the RBE increases considerably, since gamma rays produce a small number of such fragments. The analysis performed with the GBS model proved to be quite sensitive to showing, with a phenomenological single parameter, variations in double-strand break (DSB) correlation.

The proteasome inhibitor bortezomib is an antineoplastic drug mainly used for the treatment of multiple myeloma (MM). Despite its effectiveness, bortezomib clinical use is often limited by the onset of peripheral neuropathy (BiPN). To better understand the mechanisms of BiPN several rat and mice models have been proposed, but no studies in MM-bearing animals allowing to test the antitumor activity of the selected schedules and the role of MM by itself in peripheral nervous system damage have been reported to date. Here, we carried out a study using immunodeficient C.B-17/Prkdcscid (SCID) mice injected with RPMI8266 human MM cells and treated with bortezomib 1 mg/kg once a week for five weeks. Animals were assessed with neurophysiological, behavioral and pathological methods and tumor volume measurement was performed along the study. At the end of the study BiPN was evident in bortezomib-treated animals, and this neurotoxic effect was evident using a schedule able to effectively prevent tumor growth. However, neurophysiological and pathological evidence of MM induced peripheral nervous system damage was also reported. This model based on MM-bearing animals is more reliable in the reproduction of the clinical setting and it is, therefore, more suitable than the previously reported models of BiPN to study its pathogenesis. Moreover, it represents an optimal model to test the efficacy of neuroprotective agents and at the same time their non-interference with bortezomib antineoplastic activity.

Intravenous Immunoglobulin (IVIg) are human IgG derived from plasma pools of healthy donors. Although there are studies in literature evaluating their effectiveness in different pathological animal models, there are no data about their possible role on Bortezomib (BTZ)-induced peripheral neuropathies. Female Wistar rats were treated following a preventive schedule (BTZ and IVIg co-treatment for 3 and 8 weeks) and a therapeutic schedule (4 weeks of BTZ treatment followed by a 4-week IVIg-BTZ cotreatment). Caudal nerve conduction velocity (NCV), plantar and dynamic tests were performed at different time points. Animals were sacrificed after 3ws (acute phase) or 8ws (chronic phase) and tissue samples (Dorsal Root Ganglias -DRG-, sciatic nerve, caudal nerve, skin) were collected for morphological, morphometrical and immunohistological analysis.In the preventive schedule, IVIg was not able to rescue caudal NCV reduction caused by BTZ neither after 3 nor after 8 weeks of co-treatment. Same results were observed in the therapeutic schedule. On the other hand, the evaluation of mechanical allodynia and cold hyperalgesia showed that IVIg injection protected from BTZ effect in both treatment schedules. Morphometric analysis evidenced that, even if not statistically significant only the preventive schedule has a tendency to protect the caudal nerve from BTZ damage. This result is consistent with the morphological evaluation of the nerve. Also, intraepidermal nerve fibers density was preserved in the preventive schedule but not in the therapeutic one. Finally, sciatic nerve and DRG macrophage infiltration levels tended to be reduced in the therapeutic schedule and were brought back to ctrl (rats not treated or injected with IVIg alone) levels in the preventive one. In conclusion, we were able to demonstrate for the first time that IVIg treatment especially used as preventive treatment option may reduce BTZ-induced neuropathic painful pointing out the possible role of inflammation in the pathogenesis of this invalidating pathology

Aging is associated with an exaggerated response to peripheral inflammatory challenges together with behavioral and cognitive deficits. Studies considering both age and sex remain limited, despite sex dimorphism of astrocytes and microglial cells is largely recognized. To fill this knowledge gap, we investigated the effect of a single intraperitoneal lipopolysaccharide (LPS) administration in adult and aged mice. We assessed the expression of different inflammatory mediators, and the microglial response through binding of [18F]-VC701 tracer to translocator protein (TSPO) receptors in the male and female brain. Aged female brain showed a higher pro-inflammatory response to LPS compared to adult female and to aged male, as revealed by ex vivo binding to TSPO receptors and pro-inflammatory mediator transcript levels. The highest astroglial reaction was observed in the brain of aged females. Differently to the other groups of animals, in aged males LPS challenge did not affect transcription of triggering receptor expressed on myeloid cells 2 (TREM2). In conclusion, our study shows that in the mouse's brain the neuro-inflammatory response to an acute peripheral insult is sex- and age-dependent. Moreover, our results might set the basis for further studies aimed at identifying sex-related targets involved in the modulation of the aberrant neuro-inflammatory response that characterizes aging. This knowledge could be relevant for the treatment of conditions such as delirium and dementia.

Peripheral Neuropathies (PN) are common conditions whose treatment is still lacking in most cases. Animal models are crucial, but experimental procedures should be refined in some cases. We performed a detailed characterization of the ventral caudal nerve to contribute to a more effective assessment of axonal damage in future PN studies. PN was induced via weekly systemic injection of a neurotoxic drug (paclitaxel); we compared the control and PN-affected rats, performing serial neurophysiological evaluations of the caudal nerve for its entire length. On the same nerve portions, we performed light microscopy and ultrastructural pathological observations to assess the severity of damage and verify the integrity of the surrounding structures. Neurophysiological and morphological analyses confirmed that a severe axonopathy had ensued in the PN group, with a length-dependent modality, matching morphological observations. The site of neurophysiological recording (e.g., distance from the base of the tail) was critical for achieving useful data. A flexible experimental paradigm should be considered in animal studies investigating axonal PN, particularly if the expected severity is relevant; the mid-portion of the tail might be the most appropriate site: there damage might be remarkable but neither as extreme as at the tip of the tail nor as mild as at the base of the tail.

Adverse reactions, toxicity, and poor compliance from patients still represent major challenges for conventional chemotherapy treatments. Localized drug delivery would ideally improve therapeutic efficacy, minimizing the side effects. An MSU-type mesoporous silica-based nanodevice (FOL-MSN-BTZ), able to selectively deliver the antineoplastic drug bortezomib (BTZ) to folate receptor over-expressing multiple myeloma (FR+ MM) cells is described. The receptor-specific ligand, folic acid, grafted on the external surface of the nanosystem, allows tumor recognition and cell internalization, while BTZ, mainly linked to the pore internal surface through a covalent pH-sensitive bond, is released in an acidic tumor environment. A detailed investigation showed that only the fine balancing of different functionalities of the nanodevice around the external and internal surfaces of MSN particles shows the absence of toxicity towards healthy cells in vitro and negligible BTZ-release at physiological pH, which are suitable features for applicative purposes in the engineering of therapies. After complete characterization in vitro, an accurate suspendability assessment, which considered the sedimentation process that reduces the particle amount and, consequently, drug content in the suspensions, allowed the development of an injectable formulation of FOL-MSN-BTZ that showed higher antitumor efficacy and an overall tendency to lower toxicity in a MM mice model compared to the conventional bortezomib chemotherapy.

Chemotherapy-induced peripheral neurotoxicity is one of the most common dose-limiting toxicities of several widely used anticancer drugs such as platinum derivatives (cisplatin) and taxanes (paclitaxel). Several molecular mechanisms related to the onset of neurotoxicity have already been proposed, most of them having the sensory neurons of the dorsal root ganglia (DRG) and the peripheral nerve fibers as principal targets. In this study we explore chemotherapy-induced peripheral neurotoxicity beyond the neuronocentric view, investigating the changes induced by paclitaxel (PTX) and cisplatin (CDDP) on satellite glial cells (SGC) in the DRG and their crosstalk. Rats were chronically treated with PTX (10 mg/Kg, 1qwx4) or CDDP (2 mg/Kg 2qwx4) or respective vehicles. Morpho-functional analyses were performed to verify the features of drug-induced peripheral neurotoxicity. Qualitative and quantitative immunohistochemistry, 3D immunofluorescence, immunoblotting, and transmission electron microscopy analyses were also performed to detect alterations in SGCs and their interconnections. We demonstrated that PTX, but not CDDP, produces a strong activation of SGCs in the DRG, by altering their interconnections and their physical contact with sensory neurons. SGCs may act as principal actors in PTX-induced peripheral neurotoxicity, paving the way for the identification of new druggable targets for the treatment and prevention of chemotherapy-induced peripheral neurotoxicity.

Mesenchymal Stem Cells (MSCs) are adult multipotent cells able to increase sensory neuron survival: direct co-culture of MSCs with neurons is pivotal to observe a neuronal survival increase. Despite the identification of some mechanisms of action, little is known about how MSCs physically interact with neurons. The aim of this paper was to investigate and characterize the main mechanisms of interaction between MSCs and neurons. Morphological analysis showed the presence of gap junctions and tunneling nanotubes between MSCs and neurons only in direct co-cultures. Using a diffusible dye, we observed a flow from MSCs to neurons and further analysis demonstrated that MSCs donated mitochondria to neurons. Treatment of co-cultures with the gap junction blocker Carbenoxolone decreased neuronal survival, thus demonstrating the importance of gap junctions and, more in general, of cell communication for the MSC positive effect. We also investigated the role of extracellular vesicles; administration of direct co-cultures-derived vesicles was able to increase neuronal survival. In conclusion, our study demonstrates the presence and the importance of multiple routes of communication between MSCs and neurons. Such knowledge will allow a better understanding of the potential of MSCs and how to maximize their positive effect, with the final aim to provide the best protective treatment.

Chemotherapy-induced peripheral neurotoxicity is a common dose-limiting side effect of several cancer chemotherapeutic agents, and no effective therapies exist. Here we constructed a systems pharmacology model of intracellular signaling in peripheral neurons to identify novel drug targets for preventing peripheral neuropathy associated with proteasome inhibitors. Model predictions suggested the combinatorial inhibition of TNFα, NMDA receptors, and reactive oxygen species should prevent proteasome inhibitor-induced neuronal apoptosis. Dexanabinol, an inhibitor of all three targets, partially restored bortezomib-induced reduction of proximal action potential amplitude and distal nerve conduction velocity in vitro and prevented bortezomib-induced mechanical allodynia and thermal hyperalgesia in rats, including a partial recovery of intraepidermal nerve fiber density. Dexanabinol failed to restore bortezomib-induced decreases in electrophysiological endpoints in rats, and it did not compromise bortezomib anti-cancer effects in U266 multiple myeloma cells and a murine xenograft model. Owing to its favorable safety profile in humans and preclinical efficacy, dexanabinol might represent a treatment option for bortezomib-induced neuropathic pain.

Oxaliplatin (OHP)-induced peripheral neurotoxicity (OIPN) is a frequent adverse event of colorectal cancer treatment. OIPN encompasses a chronic and an acute syndrome. The latter consists of transient axonal hyperexcitability, due to unbalance in Na+ voltage-operated channels (Na+VOC). This leads to sustained depolarisation which can activate the reverse mode of the Na+/Ca2+ exchanger 2 (NCX2), resulting in toxic Ca2+ accumulation and axonal damage (ADa). We explored the role of NCX2 in in vitro and in vivo settings. Embryonic rat Dorsal Root Ganglia (DRG) organotypic cultures treated with SEA0400 (SEA), a NCX inhibitor, were used to assess neuroprotection in a proof-of-concept and pilot study to exploit NCX modulation to prevent ADa. In vivo, OHP treated mice (7 mg/Kg, i.v., once a week for 8 weeks) were compared with a vehicle-treated group (n = 12 each). Neurophysiological and behavioural testing were performed to characterise acute and chronic OIPN, and morphological analyses were performed to detect ADa. Immunohistochemistry, immunofluorescence, and western blotting (WB) analyses were also performed to demonstrate changes in NCX2 immunoreactivity and protein expression. In vitro, NCX inhibition was matched by ADa mitigation. In the in vivo part, after verifyingboth acute and chronic OIPN had ensued, we confirmed via immunohistochemistry, immunofluorescence, and WB that a significant NCX2 alteration had ensued in the OHP group. Our data suggest NCX2 involvement in ADa development, paving the way to a new line of research to prevent OIPN.

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.

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.

The development of 3D printable hydrogels based on the crosslinking between chitosan and gelatin is proposed. Chitosan and gelatin were both functionalized with methyl furan groups. Chemical modification was performed by reductive amination with methyl furfural involving the lysine residues of gelatin and the amino groups of chitosan to generate hydrogels with tailored properties. The methyl furan residues present in both polymers were exploited for efficient crosslinking via Diels-Alder ligation with PEG-Star-maleimide under cell-compatible conditions. The obtained chitosan-gelatin hybrid was employed to formulate hydrogels and 3D printable biopolymers and its processability and biocompatibility were preliminarily investigated.

Oxaliplatin is a cornerstone chemotherapeutic used in the treatment of colorectal cancer, the third leading cause of death in Western countries. Most side effects of this platinum-containing drug are adequately managed in the clinic, although acute and long-term neurotoxicity still severely compromises the quality of life of patients treated with oxaliplatin. We have previously demonstrated that therapeutically relevant concentrations/doses of oxaliplatin lead to a reduction in intracellular pH in mouse dorsal root ganglion (DRG) neurons in vitro and in vivo and that this alteration sensitizes TRPA1 and TRPV1 channels, which most likely mediate the allodynia associated with treatment. In this study, we show that oxaliplatin leads to a reduction of intracellular pH by forming adducts with neuronal haemoglobin, which acts in this setting as a proton buffer. Furthermore, we show that FDA-approved drugs that inhibit carbonic anhydrase (an enzyme that is linked to haemoglobin in intracellular pH homeostasis), ie, topiramate and acetazolamide, revert (1) oxaliplatin-induced cytosolic acidification and TRPA1 and TRPV1 modulation in DRG neurons in culture, (2) oxaliplatin-induced cytosolic acidification of DRG of treated animals, and (3) oxaliplatin-induced acute cold allodynia in mice while not affecting OHP-induced cytotoxicity on cancer cells. Our data would therefore suggest that reversal of oxaliplatin-induced cytosolic acidification is a viable strategy to minimize acute oxaliplatin-induced symptoms.

Oxaliplatin (OHP) Induced Peripheral Neurotoxicity (OIPN) is one of the dose-limiting toxicities of the drug and these adverse effects limit cancer therapy with L-OHP, used for colorectal cancer treatment. Acute neurotoxicity consists of symptoms that are the hallmarks of a transient axonal hyperexcitability; chronic neurotoxicity has a clinical picture compatible with a length-dependent sensory neuropathy. Acute OIPN pathogenesis has been linked to sodium voltage-operated channels (Na + VOC) dysfunction and it has been advocated as a possible predisposing factor to chronic neurotoxicity. We tested if topiramate (TPM), a well-known Na + VOC modulator, was able to modify acute as well as chronic OIPN. The project was divided into two parts. In Experiment 1 we tested by means of Nerve Excitability Testing (NET) a cohort of female Wistar rats to assess TPM effects after a single OHP administration (5 mg/kg, iv). In Experiment 2 we assessed TPM effects after chronic OHP treatment (5 mg/kg, 2qw4ws, iv) using NET, nerve conduction studies (NCS), behavioral tests and neuropathology (caudal nerve morphometry and morphology and Intraepidermal Nerve Fiber [IENF] density). In Experiment 1 TPM was able to prevent OHP effects on Na + VOC: OHP treatment induced a highly significant reduction of the sensory nerve's threshold, during the superexcitability period (p-value = 0.008), whereas TPM co-administration prevented this effect. In Experiment 2 we verified that TPM was able to prevent not only acute phenomena, but also to completely prevent chronic OIPN. This latter observation was supported by a multimodal approach: in fact, only OHP group showed altered findings compared to CTRL group at a neurophysiological (proximal caudal nerve sensory nerve action potential [SNAP] amplitude, p-value = 0.001; distal caudal nerve SNAP amplitude, p-value

Celiac disease (CD) is an autoimmune enteropathy arising in genetically predisposed subjects exposed to gluten, which activates both innate and adaptive immunity. Although the pathogenesis is common to all patients, the clinical spectrum is quite variable, and differences could be explained by gene expression variations. Among the factors able to affect gene expression, there are lncRNAs. We evaluated the expression profile of 87 lncRNAs in CD vs. healthy control (HC) intestinal biopsies by RT-qPCR array. Nuclear enriched abundant transcript 1 (NEAT1) and taurine upregulated gene 1 (TUG1) were detected as downregulated in CD patients at diagnosis, but their expression increased in biopsies of patients on a gluten-free diet (GFD) exposed to gluten. The increase in NEAT1 expression after gluten exposure was mediated by IL-15 and STAT3 activation and binding to the NEAT1 promoter, as demonstrated by gel shift assay. NEAT1 is localized in the nucleus and can regulate gene expression by sequestering transcription factors, and it has been implicated in immune regulation and control of cell proliferation. The demonstration of its regulation by gluten thus also supports the role of lncRNAs in CD and prompts further research on these RNAs as gene expression regulators.

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 Chemotherapy-induced peripheral neurotoxicity (CIPN) is a severe adverse effect in patients receiving antitumor agents, and no effective treatment is available. Although the mechanisms responsible for the development of CIPN are poorly understood, recent findings make neuroinflammation an attractive target to be investigated, particularly when neuropathic pain is a prominent feature such as after bortezomib administration. The aim of our study was to evaluate the effect of intravenous immunoglobulins (IVIg) delivery in chronic CIPN. The related neuro-immune aspects were investigated in a well-characterized rat model of bortezomib-induced peripheral neurotoxicity (BIPN). Methods After determination of a suitable schedule based on a preliminary pharmacokinetic pilot study, female Wistar rats were treated with IVIg 1 g/kg every 2 weeks. IVIg treatment was started at the beginning of bortezomib administration (“preventive” schedule), or once BIPN was already ensued after 4 weeks of treatment (“therapeutic” schedule). Neurophysiological and behavioral studies were performed to assess the extent of painful peripheral neurotoxicity induced by bortezomib, and these functional assessments were completed by pathologic examination of peripheral nerves and intraepidermal nerve fiber quantification (IENF). The role of the innate immune response in BIPN was investigated by immunochemistry characterization of macrophage infiltration in peripheral nerves. Results Both schedules of IVIg administration were able to significantly reduce bortezomib-induced heat and mechanical allodynia. Although these changes were not evidenced at the neurophysiological examination of peripheral nerves, they behavioral effects were paralleled in the animals treated with the preventive schedule by reduced axonopathy in peripheral nerves and significant protection from loss of IENF. Moreover, IVIg administration was very effective in reducing infiltration in peripheral nerves of macrophages with the M1, pro-inflammatory phenotype. Conclusion Our results suggest a prominent role of neuroinflammation in BIPN and that IVIg might be considered as a possible safe and effective therapeutic option preventing M1 macrophage infiltration. However, since neuropathic pain is frequent also in other CIPN types, it also indicates the need for further investigation in other forms of CIPN.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major cause of disability in cancer survivors. CIPN investigations in preclinical model systems have focused on either behaviors or acute changes in nerve conduction velocity (NCV) and amplitude, but greater understanding of the underlying nature of axonal injury and its long-term processes is needed as cancer patients live longer. In this study, we used multiple independent endpoints to systematically characterize CIPN recovery in mice exposed to the antitubulin cancer drugs eribulin, ixabepilone, paclitaxel, or vinorelbine at MTDs. All of the drugs ablated intraepidermal nerve fibers and produced axonopathy, with a secondary disruption in myelin structure within 2 weeks of drug administration. In addition, all of the drugs reduced sensory NCV and amplitude, with greater deficits after paclitaxel and lesser deficits after ixabepilone. These effects correlated with degeneration in dorsal root ganglia (DRG) and sciatic nerve and abundance of Schwann cells. Although most injuries were fully reversible after 3–6 months after administration of eribulin, vinorelbine, and ixabepilone, we observed delayed recovery after paclitaxel that produced a more severe, pervasive, and prolonged neurotoxicity. Compared with other agents, paclitaxel also displayed a unique prolonged exposure in sciatic nerve and DRG. The most sensitive indicator of toxicity was axonopathy and secondary myelin changes accompanied by a reduction in intraepidermal nerve fiber density. Taken together, our findings suggest that intraepidermal nerve fiber density and changes in NCV and amplitude might provide measures of axonal injury to guide clinical practice. Significance: This detailed preclinical study of the long-term effects of widely used antitubulin cancer drugs on the peripheral nervous system may help guide clinical evaluations to improve personalized care in limiting neurotoxicity in cancer survivors. Cancer Res; 78(3); 817–29. ©2017 AACR.