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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.

Mesenchymal stem cells (MSCs) have been often proposed for the therapy of several neurological diseases, due to their manifold peculiar properties. In particular, since it has been previously demonstrated that these cells are able to increase the survival of untreated sensory neurons [1], in this work we evaluated their possible protective effect on sensory neurons previously exposed to toxic agents. This could be particularly relevant to design a supportive therapy to counteract the peripheral neuropathy, a very common side effect of several chemotherapeutic agents, such as platinum and taxanes compounds, which often represents their dose limiting factor [2]. Several strategies have been suggested to reduce drug neurotoxicity without affecting the antineoplastic potential, but up to now results were not encouraging [3]. Here we demonstrated that Cisplatin (CDDP) and Paclitaxel-treated sensory neurons are protected by the co-culture with MSCs, but in two different manners: through a direct contact able to block apoptosis for CDDP-treated neurons, and by the release of trophic factors (including glutathione) for Paclitaxel-treated ones. In addition, the MSCs’ effectiveness was also verified on cortical neurons, since the recent advances in targeted drug delivery allowed to drive chemotherapeutic drugs also to the central nervous system. We verified that cortical neurons are more vulnerable to the toxic action of the drugs, and overall that MSCs fail at all to protect them. All these data demonstrated that MSCs are potentially useful to limit the peripheral neuropathy onset for their protective effect on injured-sensory neurons, but they also identified for the first time a different susceptibility of cortical and sensory neurons to MSC action., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

Axonal transport of mitochondria (Mt) controlled by specialized motor and docking proteins that distribute Mt throughout the axon where they provide energy for metabolic and synaptic activity is a vulnerable target in neuronal pathology (1). Bortezomib (BZ) is a proteasome inhibitor active in multiple myeloma (2). One of its key toxicities is painful peripheral neuropathy (BIPN), which frequently requires treatment discontinuation (3). BIPN is dose-related and predominantly sensory, resulting from axonal degeneration. Recent results indicate that BZ modifies axonal tubulin dynamic and we hypothesize that BZ alters fast axonal transport. Here we studied using time-lapse imaging the effect of different BZ concentration on axonal Mt transport in isolated dorsal root ganglion (DRG) neurons from adult male mice. We used kymograph to quantify the total number of Mt and to discriminate antero and retrogradely moving Mt from stationary Mt. Twenty-four hours of BZ treatment (0.1 to 15 µM) induced a dose-dependent reduction in Mt trafficking. Moreover, BZ had no impact on MT motion directions, but it induced a progressive reduction of both anterograde and retrograde axonal transport velocities. These events were associated with increase in tubulin polymerization and of MAP2 expression, but they occurred only after 72h of chronic BZ treatment. We have developed an in vitro model of BIPN demonstrating that transport impairment is already present before evident tubulin polymerization, suggesting that transport deficit represents an early stage of axonal dysfunction. Perpetuated transport dysfunction could impair distal organelle supply and play a critical role in advanced stages of BIPN.This work was supported by the University of Milan-Bicocca and University of Michigan research grants, Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

A step forward in the identification of compounds interfering with the Embryonic Lethal Abnormal Vision (ELAV) protein - RNA complexes

Sigma 1 receptor modulators as a new weapon against multiple sclerosis

Oxygen and glucose deprivation (OGD) due to ischemic events or trauma in the brain result in neuronal loss. The therapeutic approaches available inadequate and often the outcome is unfavorable for the patient or at least unpredictable. Stem cells could be useful for the treatment of OGD injured-neurons. Mesenchymal Stem Cells (MSCs), isolated from bone marrow as well as from various tissues, have poor immunogenicity and neuroprotective properties being able to alleviate ischemic brain injuries in animal models. The Endothelial Progenitor Cells (EPCs) are present at low frequencies both in the bone marrow and in the peripheral blood. They are thought to play a role in the recovery of cerebrovasculature integrity after stroke. In the present study we evaluated the potential neuroprotective effect of human MSCs and human EPCs on rat embryonic cortical neurons injured by OGD. OGD was induced by incubating the cortical neurons in a hypoxia chamber in a 95% N2 + 5% CO2 atmosphere at 37°C without glucose. To set up the experimental protocol, OGD was maintained for 1, 2 and 3 hours. The neurons were returned in normoxic atmosphere and after 2 and 5 days neuronal survival was evaluated by MTT assay, LDH assay and viable cellular counting. The 2 hours OGD was able to reduce neuronal viability by 50% and was chosen for the subsequent experiments. To assess MSCs and EPCs neuroprotective action, after 2 hours-long OGD the neurons were 1) co-cultured with either MSCs or EPCs seeded on a cell culture insert avoiding direct contact while sharing the same medium, or 2) cultured in a medium previously conditioned by either MSCs or EPCs. Neuronal survival was evaluated by MTT assay after 2 and 5 days. Both MSCs and EPCs increased neuronal survival after ODG. The effect was observed in absence of a direct contact between MSCs or EPCs and the injured neurons, suggesting that the release of soluble factors may be involved in their neuroprotective action. In conclusion both MSCs and EPCs could represent a potential therapeutic approach for the treatment of brain ischemic injury. Further studies are needed to identify the specific molecules and pathways that play a role in the neuroprotective effect of MSCs and EPCs., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

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

Treatment of type-1diabetes with pancreatic islet transplantation is an intriguing therapeutic option, aimed to replace insulin administration, but very limited in clinical practice, mainly for the great number of islets necessary and for their short survival. Aim of this work is to verify the ability of Mesenchymal Stem Cells (MSCs) co-transplanted with Pancreatic Islets to improve the feasibility of this approach, by acting both on glycaemic control and on long term disease complications, such as the diabetic neuropathy. 5 groups were used (8 rats/group): a) healthy controls; b) Streptozotocin-induced diabetic rats; c) Diabetic rats transplanted with pancreatic islets (3000); d) Diabetic rats co-transplanted with pancreatic islets (2000) and MSCs (106); Diabetic rats treated with MSCs (106). Transplantations were performed after the assessment of neuropathic signs, such as the decrease of Nerve Conduction Velocity (NCV) and the 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 marked and significant glycaemia value reduction, an improvement of thermal and mechanical sensitivity, and a nearly complete restoration of NCV with respect to diabetic-untreated rats. No differences were observed between diabetic rats and diabetic rats treated with only MSCs. Co-transplantation of MSCs with Pancreatic Islets allows to reduce the successful number of pancreatic islets, to obtain a better and more physiologic glycaemic control, and to induce the regression of painful neuropathy signs, thus ameliorating diabetes complications management

Neurodegenerative diseases are different and many-sided disorders affecting both the Central and the Peripheral Nervous System. Despite the very different peculiar features, all the neurodegenerative diseases are characterized by the neuronal degeneration, which may be the consequence of different processes, such as an altered protein accumulation, an axonal damage, or the exposure to toxic agents. The progressive neuronal death leads to disease progression, which is not effectively counteracted by the current symptomatic therapies. Among the newly proposed therapeutic approaches, encouraging results have been obtained with Mesenchymal Stem Cells (MSCs), adult stem cells initially proposed for their differentiation potential and for their immune-modulatory abilities. Here we first verified in vivo the protective potential of MSCs into an in vivo model of Multiple Sclerosis (MS), represented by Experimental Autoimmune Encephalomyelitis (EAE), demonstrating that intravenous administration of MSCs are able to ameliorate the clinical score and the functional skills, and to reduce demyelinated lesions. We then investigated in vitro the possible molecular mechanisms of MSC protective action, thus demonstrating that, besides immunomodulation, MSCs are able to support neuronal survival after toxic stimuli exposure by reducing the apoptosis and by inhibiting the Metalloprotease pathway, which is supposed to be involved in neurodegenerative disease progression. Moreover, MSCs are able to promote the axonal myelination through the modulation of p75 receptor. For all these abilities, MSCs can represent a promising therapeutic approach for the treatment of neurodegenerative disorders.

Treatment of type-1diabetes with pancreatic islet transplantation is an innovative therapeutic approach, but itsclinical application is still very limited, mainly for the great number of islets necessary and for their short survival. An intriguing means to improve the performance of pancreatic islets transplantation may be represented by Mesenchymal Stem Cells (MSCs), adult stem cells already known to support the survivalof different cellular populations. In this work we investigated the ability of Mesenchymal Stem Cells (MSCs) to reduce the number of pancreatic islets necessary to achieve glycaemic control in Streptozotocin-induced diabetic rats, and their effect ona long term disease complication,the diabetic neuropathy. Diabetic rats showed high blood glucose level and important neuropathic signs, such as the decrease of Nerve Conduction Velocity (NCV) and the impairment of nociceptive (thermal and mechanical) thresholds. Two months after transplantation, diabetic rats co-transplanted with MSCs and a suboptimal number of pancreatic islets showed a marked and significant glycaemia value reduction, an improvement of thermal and mechanical sensitivity, and a nearly complete restoration of NCV with respect to diabetic-untreatedrats. The in vitro analysis of the putative mechanisms of MSC positive action on pancreatic islets suggested the involvement of both trophic soluble factors released by MSCs, and their differentiation into insulin-releasing cells after the direct contact with pancreatic islets. In conclusion, we demonstrated that co-transplantation with MSCs reduces the number of pancreatic islets needed to reach glycaemic control, and inducesthe regression of painful neuropathy signs, thus ameliorating diabetes complicationsmanagement.

The cell-based therapy is a promising approach to treat many degenerative diseases such as type 1 Diabetes Mellitus (T1DM). Besides the exclusive pharmacological treatment for T1DM a new approach has been recently proposed for restoring of Beta cell mass by islet transplantation. One of the principal problems of this approach is the numerical and functional loss of transplanted islets. For these reasons new strategies are studied in order to increase islets survival. In our laboratories we have already demonstrated that rat Mesenchymal Stem Cells (rMSC) are able to promote islets survival in vitro and that rMSC, if cocultured with pancreatic islets, are able to express Pdx1, a gene involved in beta cell insulin secretion. The aim of this study is to verify the effect of human Mesenchymal Stem Cells (hMSC) on the survival and function of pancreatic islets. In order to clarify which mechanism could be involved in the putative positive effect we set up different culture conditions: direct coculture, in which hMSC were in direct contact with islets; indirect coculture in which hMSC and islets shared the medium; mix cocultured in which islets were both in direct contact and shared the medium with hMSC. Preliminary results demonstrate a positive effect of hMSC on islets survival. Now we are focusing on the effect on insulin secretion regulated by hMSC in the different coculture conditions.

There are promising studies which demonstrate that Mesenchymal Stem Cells (MSCs) are able to release neutrophic factors, to increase neuronal survival and as a consequence to repair nervous damages. For these reasons MSCs based therapeutic approach was proposed for diseases of both central and peripheral nervous system. In order to evaluate a putative positive effect of MSCs on neuronal damage recovery, two different experimental models were set up using cortical and sensory neurons. The toxic stimuli used were: Glutamate, whose toxicity is associated to Multiple Sclerosis; Cisplatin and Paclitaxel, chemotherapic drugs, which induce peripheral neuropathies. For the evaluation of the effect of MSCs, direct and indirect co-cultures were set up with rat MSCs (rMSCs); rMSCs Conditioned Medium effect was also evaluated. Our results demonstrate an important difference between cortical and sensory neurons in fact in term of susceptibility cortical neurons are much more sensible to drugs treatment. About sensory neurons we observed a protective action in direct co-cultures on Cisplatin and Glutamate treated neurons and in indirect co-cultures on Paclitaxel treated neurons; conditioned medium didn’t have any protective action. However direct and indirect co-cultures and condition medium were unable to protect cortical neurons form toxic drugs. These data could indicate that a possible MSCs based therapy would be more promising for treating peripheral nervous system diseases rather than central one.