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The development and progression of diabetic polyneuropathy (DPN) are due to multiple mechanisms. The creation of reliable animal models of DPN has been challenging and this issue has not yet been solved. However, despite some recognized differences from humans, most of the current knowledge on the pathogenesis of DPN relies on results achieved using rodent animal models. The simplest experimental DPN model reproduces type 1 diabetes, induced by massive chemical destruction of pancreatic beta cells with streptozotocin (STZ). Spontaneous/transgenic models of diabetes are less frequently used, mostly because they are less predictable in clinical course, more expensive, and require a variable time to achieve homogeneous metabolic conditions. Among them, Zucker diabetic fatty (ZDF) rats represent a typical type 2 diabetes model. Both STZ-induced and ZDF rats have been extensively used, but only very few studies have compared the long-term similarities and differences existing between these two models. Moreover, inconsistencies have been reported regarding several aspects of short-term in vivo studies using these models. In this study, we compared the long-term course of DPN in STZ-treated Sprague–Dawley and ZDF rats with a multimodal set of readout measures.

In this study, we investigate the occurrence and magnitude of plastic pollution in the Sub-Arctic ecosystem through the monitoring of several indicative variables in a sentinel organism, the fin whale (Balaenoptera physalus) from the waters off western Iceland. We followed two approaches. The first was the analysis of microplastics in fin whale’s stomach content (n=25, collected in 2018). The second was the analysis of two sets of plastic additives: phthalates in fin whale muscle (n=31, collected along the period 1986-2015), and organophosphate flame retardants (OPFR) in both fin whale muscle (n=20, collected in 2015) and stomach contents (n=10, collected in 2009-2013). We found that the stomach contents, which were composed of krill (Meganyctiphanes norvegica), the mean concentration of microplastics was 0.057 particles per gram of krill, suggesting a fin whale daily intake of 38,646 to 77,292 particles per day. Regarding phthalates, we found that the most abundant compounds were: di-n-butylphthalate (DBP), diethylphthalate (DEP), and bis(2-ethylhexyl) phthalate (DEHP). We did not observe any significant trend in the phthalate concentrations over the 29-year period studied. Regarding organophosphate flame retardants (OPFR) we found the occurrence of seven OPFRs compounds in the fin whale muscle and five in krill, being tri-n-butyl phosphate (TNBP), Isopropylated triphenyl phosphate (IPPP), and Triphenylphosphine oxide (TPPO) the most abundant compounds in both matrices. Our results also support the absence of OPFR krill-to-whale biomagnification as well as the bioaccumulation of these compounds in fin whale tissues. These findings highlight the need for continued monitoring of plastic pollution and its associated chemicals in marine wildlife, particularly in large filter-feeding species such as the fin whale.

Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB.

Adaptation to life in the deep-sea can be dramatic, with fish displaying behaviors and appearances unlike those seen in any other aquatic habitat. However, the extent of which adaptations may have developed at a microbial scale is not as clear. Shotgun metagenomic sequencing of the intestinal microbiome of 32 species of deep-sea fish from across the Atlantic Ocean revealed that many of the associated microbes differ extensively from those previously identified in reference databases. 111 individual metagenome-assembled genomes (MAGs) were constructed representing individual microbial species from the microbiomes of these fish, many of which are potentially novel bacterial taxa and provide a window into the microbial diversity in this underexplored environment. These MAGs also demonstrate how these microbes have adapted to deep-sea life by encoding a greater capacity for several cellular processes such as protein folding and DNA replication that can be inhibited by high pressure. Another intriguing feature was the almost complete lack of genes responsible for acquired resistance to known antibiotics in many of the samples. This highlights that deep-sea fish microbiomes may represent one of few animal-associated microbiomes with little influence from human activity. The ability of the microbes in these samples to bioluminesce is lower than expected given predictions that this trait has an important role in their life cycle at these depths. The study highlights the uniqueness, complexity and adaptation of microbial communities living in one of the largest and harshest environments on Earth.

Adaptation to life in the deep-sea can be dramatic, with fish displaying behaviors and appearances unlike those seen in any other aquatic habitat. However, the extent of which adaptations may have developed at a microbial scale is not as clear. Shotgun metagenomic sequencing of the intestinal microbiome of 32 species of deep-sea fish from across the Atlantic Ocean revealed that many of the associated microbes differ extensively from those previously identified in reference databases. 111 individual metagenome-assembled genomes (MAGs) were constructed representing individual microbial species from the microbiomes of these fish, many of which are potentially novel bacterial taxa and provide a window into the microbial diversity in this underexplored environment. These MAGs also demonstrate how these microbes have adapted to deep-sea life by encoding a greater capacity for several cellular processes such as protein folding and DNA replication that can be inhibited by high pressure. Another intriguing feature was the almost complete lack of genes responsible for acquired resistance to known antibiotics in many of the samples. This highlights that deep-sea fish microbiomes may represent one of few animal-associated microbiomes with little influence from human activity. The ability of the microbes in these samples to bioluminesce is lower than expected given predictions that this trait has an important role in their life cycle at these depths. The study highlights the uniqueness, complexity and adaptation of microbial communities living in one of the largest and harshest environments on Earth.

The diversity of Phytophthora species in declining Fagaceae forests in Europe is increasing in the last years. The genus Quercus is one of the most extended Fagaceae genera in Europe, and Q. ilex is the dominant tree in Spain. The introduction of soil-borne pathogens, such as Phytophthora in Fagaceae forests modifies the microbial community present in the rhizosphere, and has relevant environmental and economic consequences. A better understanding of the diversity of Phytophthora spp. associated with Q. ilex is proposed in this study by using Next Generation Sequencing (NGS) in six Q. ilex stands located in three regions in Spain. Thirty-seven Phytophthora phylotypes belonging to clades 1 to 12, except for clades 4, 5 and 11, are detected in this study, which represents a high diversity of Phytophthora species in holm oak Spanish forests. Phytophthora chlamydospora, P. citrophthora, P. gonapodyides, P. lacustris, P. meadii, P. plurivora, P. pseudocryptogea, P. psychrophila and P. quercina were present in the three regions. Seven phylotypes could not be associated with known Phytophthora species, so they were putatively named as Phytophthora sp. Most of the detected phylotypes corresponded to terrestrial Phytophthora species but aquatic species from clades 6 and 9 were also present in all regions.

The occurrence of classical and emerging halogenated flame retardants in bird samples collected between 2010–17 from the Castrejón reservoir (central Spain)was studied. Different wetland bird samples were analysed, including unhatched bird eggs and liver of dead nestlings. Polybrominated diphenylethers (PBDEs)were detected in all the samples at high concentration values, with levels up to 5167 ng/g lw. Dechloranes were found in 78% of analysed samples, but at lower concentration levels, between not detected (nd)and 2153 ng/g lw. The time trend evaluation over the sampling period showed an approximately 50% decline in mean concentrations of PBDEs. However, the most recent data for PBDEs (2016–17)still indicate that, in some cases, and based on reported LOECs, wetland birds were exposed to PBDE concentrations that are associated with possible ecological hazards. © 2019 Elsevier Ltd

PBDEs, HBCD, DBDPE, PBEB and HBB, dechloranes and OPFRs, as well as natural MeO-PBDEs were monitored in muscle tissue of three dolphin species from the southwestern Indian Ocean (Delphinus delphis, Sousa plumbea and Tursiops aduncus) collected between 2012 and 2015. The mean PBDE concentration was 416 ± 333 ng g −1 lw. BDE-47 was found in all samples and was almost half the total PBDE contamination. BDE-209, BDE-100 and BDE-99 were present in ≥85% of the samples. HBCD was detected in just two samples at 20 and 330 ng g −1 lw. PBEB and HBB were not detected, while DBDPE was in all samples but always below its limit of quantification. Dec 602 was the only quantifiable dechlorane at 232 ± 549 ng g −1 lw. Mean OPFR concentration was 10452 ± 11301 ng g −1 lw. TBOEP was found in all samples making up most of the total OPFR contamination. MeO-PBDEs were detected in all samples at 114 ± 137 ng g −1 lw. Data on flame retardants in biota and environmental samples from the southwestern Indian Ocean are scarce and, as a result, comparisons are difficult. However, data from other marine predators in the region, such as penguins, suggest that further studies are needed to determine if these concentrations are the consequence of a high local contamination or widespread thoughout the Indian Ocean. © 2019 Elsevier Ltd

Aims: Mitochondria are very dynamic organelles. Mitochondrial dynamics (alternation of fission/fusion cycles and their axonal transport) and biogenesis are crucial for neuronal homeostasis and synaptic function; moreover, alteration of the balance between fission and fusion can determine the fate of neuronal cells. Fission processes require the intervention of Fis1 and the active form of Drp1, whereas fusion is primarily regulated by Opa1 and Mfn1/2. We here focused on correlation between oxidative stress and mitochondrial dynamics and biogenesis. Materials and Methods: We compared the effect of hydrogen peroxide and Rotenone (a neurotoxin that inhibits the Complex-I of electrons transport chain), on primary cortical neurons or models of neurodegeneration based on neuroblastoma or PC12 cells. Protein levels and distribution (cytoplasm, mitochondria or nuclei) were assessed by western blots and immunofluorescence imaging. Results: Exposure of neurons or neuroblastoma to Rotenone determined a dose-dependent (200-400-800 nM) decrease of P-Drp1 and a biphasic trend of Opa1 and Mfn2 in a time-dependent manner (1-3-6-24h). In PC12 cells, alterations of mitochondrial proteins in response to hydrogen peroxide was paralleled by differential distribution (cytoplasm/nuclei) of oxidative-stress sensors (DJ-1, Nrf2) and proteins regulating mitochondrial biogenesis (mtTFAM and PGC1a), while determining a dose- and time-dependent increase of ROS and ATP depletion. Interestingly, human fibroblasts showed similar changes in response to rotenone but a different trend in response to Amyloid-beta oligomers. Discussion/Conclusions: These studies suggest 1) the existence of a complex cross-talk underlying mitochondrial dynamics and biogenesis, 2) alteration of mitochondrial dynamic proteins as biomarkers of mitochondrial dysfunction in neurodegenerative processes.

La incidencia de la enfermedad cardiovascular (ECV) se ha incrementado en los pacientes infectados por el VIH. La replicación del VIH y la terapia antirretroviral pueden contribuir de forma independiente al aumento del riesgo de ECV. Sin embargo, la causa de este aumento del riesgo es multifactorial. Métodos: Esta tesis se centra en el estudio de los trastornos metabólicos y cardiovasculares en una cohorte de pacientes infectados por el VIH. Para ello, se llevaron a cabo tres estudios: Un primer estudio, diseñado con el propósito de determinar si un análisis combinado de variantes genéticas en algunos de los genes responsables de las anomalías lipídicas ayudarían a discriminar los pacientes infectados por el VIH con niveles altos de triglicéridos y bajos niveles de HDL-colesterol (pacientes con perfil aterogénico), considerado un marcador claro de riesgo cardiovascular. Para ello, se realizó la caracterización genética y metabólica de 321 pacientes infectados por el VIH en tratamiento antirretroviral, a través de la determinación del perfil lipídico, la determinación de partículas remanentes de colesterol y la determinación de los genes implicados en la formación y en la eliminación de partículas remanentes de la circulación. Se identificaron varios genes responsables de la modulación de los parámetros lipídicos en esta población. Sin embargo, el diseño transversal del estudio y el pequeño tamaño de la muestra no permitió conclusiones definitivas acerca de la asociación entre polimorfismos en los genes asociados con el metabolismo de los lípidos y los trastornos de lípidos en pacientes infectados por el VIH. El segundo estudio, se centró en la detección precoz de la aterosclerosis subclínica. Para ello, se evaluó la elasticidad arterial a través de la determinación de la velocidad de la onda de pulso (VOP) en pacientes infectados por el VIH, en comparación con sujetos no infectados. Se identificaron factores de riesgo asociados con la alteración de la elasticidad ar, The incidence of cardiovascular disease (CVD) has increased among HIV-infected patients. Both HIV replication and antiretroviral therapy may contribute independently to increase the risk of CVD. However, the cause of this increased risk is multifactorial. Methods: This thesis focuses on the study of metabolic and cardiovascular disorders in a cohort of HIV-infected patients. Three studies were conducted: A first study was designed with the purpose of ascertaining whether a combined analysis of several genetic variants in some of the genes responsible of lipid abnormalities helped discriminate HIV-infected patients with high triglycerides and low levels of HDL-cholesterol (patients with atherogenic profile), clear marker of cardiovascular risk. It was carried out metabolic and genetic characterization of 321 HIV-infected patients receiving antiretroviral therapy, through the determination of the lipid profile, determination of remnant cholesterol particles and determination of genes involved in the formation, as in removing remaining particles from circulation. Several genes responsible for modulation of lipid parameters in this population were identified. However, the cross-sectional design of the study and the small sample size does not allow definitive conclusions about the association between polymorphisms in genes associated with lipid metabolism and lipid disorders in HIV-infected patients. The second study focused on the early detection of subclinical atherosclerosis. For this, arterial elasticity was evaluated through the determination of the pulse wave velocity (PWV) in HIV-infected patients, compared to non-infected subjects. Risk factors associated with impaired arterial compliance were identified. HIV-infected patients with virologic suppression showed similar arterial elasticity than non-infected subjects. HIV-related conditions were not associated with arterial stiffness. However, high diastolic pressure at the time of the PWV and high triglycerides wer

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.

In this work, genes encoding gelatinase (gelE) and serine proteinase (sprE), two extracellular proteases produced by Enterococcus faecalis DBH18, were cloned in the protein expression vector pMG36c, containing the constitutive P32 promoter, generating the recombinant plasmids pCG, pCSP, and pCGSP encoding gelE, sprE, and gelE-sprE, respectively. Transformation of noncaseinolytic E. faecalis P36, E. faecalis JH2-2, E. faecium AR24, and E. hirae AR14 strains with these plasmids permitted detection of caseinolytic activity only in the strains transformed with pCG or pCGSP. Complementation of a deletion (knockout) mutant of E. faecalis V583 for production of gelatinase (GelE) with pCG unequivocally supported that gelE is responsible for the caseinolytic activity of the transformed strain grown in bovine skim milk (BSM). RP-HPLC-MS/MS analysis of hydrolysates of transformed Enterococcus spp. strains grown in BSM permitted the identification of 38 major peptide fragments including peptides with previously reported angiotensin converting enzyme-inhibitory activity (ACE-IA), antihypertensive activity, and antioxidant activity. © 2014 American Chemical Society.

Cloning of the enterolysin A (EnlA) gene (enlA) from Enterococcus faecalis DAC9 into the pMSP3545-derived pMLG2-protein expression vector encoding EnlA under control of the inducible PnisA promoter permitted the controlled release and heterologous expression of mature EnlA by Lactococcus lactis subsp. cremoris NZ9000 and Lactococcus lactis subsp. lactis IL1403. The nisin-induced expression of enlA by L.lactis NZ9000 (pMLG2) and L.lactis IL1403 (pMLG2), grown in GM17 or bovine skim milk (BSM), caused a noticeable reduction of the optical density (OD600) of the cultures and death of the growing cells. However, a high angiotensin converting enzyme (ACE)-inhibitory activity (ACE-IA) was only observed in the BSM-derived hydrolysates of L.lactis IL1403 (pMLG2) after 48h-induction with nisin. Analysis of these hydrolysates by reversed phase high performance liquid chromatography-tandem mass spectrometry permitted the identification of major peptide fragments with known ACE-IA or sharing at least three C-terminal residues with those displaying ACE-IA. © 2013 Elsevier Ltd.

Cisplatin is an anticancer drug employed for the treatment of several solid tumours, including testicular, ovarian, breast and lung cancers. However, treatment side effects remain an unsolved problem. Some of the patients treated with cisplatin, after a cumulative dose of 300 mg/m2, develop a painful peripheral neuropathy (PPN) characterized by distal paresthesias, numbness, sensory ataxia and neuropathic pain. Since a different degree of the PPN severity suggests an individual variation in the drug toxicity response, the understanding of the genomics underlying PPN would lead to new therapeutic targets and to early diagnostic screening. In this study we assessed the severity of cisplatin-induced PPN in six mouse strains using Nerve Conduction Velocities (NCV), Neurometer studies, morphological analysis of DRG, caudal and sciatic nerves and lumbar spinal cord, morphometrical analysis of DRG and behavioral assessment of mechanical allodynia. Cisplatin 4 mg/Kg was intraperitoneally administered twice a week for 4 weeks in CD1, Balb-c, C57BL6, FVB, DBA and AJ mice. Cisplatin induced a significant impairment of caudal and digital NCV in all except FVB and DBA mice. Moreover, cisplatin administration resulted in a significant decrease in the response to mechanical stimulation in Balb-c, C57BL6, CD1 and DBA starting from the first week of treatment while AJ and FVB never develop mechanical allodynia. Neurometer analysis, that allows a quantitative assessment of the functionality of the three major subpopulations of sensory nerve fibers (A-delta, A-beta and C), showed that large myelinated fibers (A-beta) were affected by cisplatin in Balb-c and CD1, small myelinated (A-delta) only in Balb-c while small unmyelinated fibers (C) were unaffected only in FVB and AJ mice. The morphometrical analysis revealed a nucleolar and somatic atrophy of DRG sensory neurons of CD1 but not of FVB-treated mice. Taken together, these results demonstrate that CD1 and FVB mice were the most and

Bortezomib (BTZ) is a proteasome inhibitor used as first-line therapy for multiple myeloma. However, its administration induces the development of severe painful peripheral neuropathy (PPN). This painful condition is an important medical need since the available treatments are actually ineffective. We recently described a mice model of PPN that shares most of the conditions found in patients treated chronically with BTZ (Carozzi et al., 2013). In fact, BTZ determines dysfunction of all fiber types in sensory nerves and, at least in mice, alters the electrical activity of the spinal dorsal horn neurons. This alteration of the basal electrophysiological activity induces also relevant changes in the central nociceptive transmission. In this work we characterize the neuroprotective effects of an imidazoline receptor 2 ligand (CR4056) able to allosterically inhibit the activity of monoamine oxidase-A, a key enzyme in the regulation of neuropathic pain. Wistar rats were treated with BTZ 0.20 mg/kg, 3 times a week for 8 weeks (i.v). Then CR4056 was orally administered in a curative schedule at 6 mg/kg, once a day, for 2 weeks. Gabapentin (100 mg/kg, daily, p.o.) and buprenorphine (28,8µg/kg, daily, s.c.) were used as internal analgesic standards. At the end of both BTZ and analgesic treatments, we measured the caudal and sciatic nerve conduction velocity (NCV), the morphological/morphometrical alterations in the caudal nerve and the neuropathic pain development. BTZ treatment induced a significant impairment of sensory, but not motor NCV, slight hyperalgesia, significant mechanical allodynia and clearing of myelinated fibers in the caudal nerves. After two weeks of follow up animals did not spontaneously recover functional, morphological and behavioral abnormalities while the 2 weeks-treatment with CR4056 (but not with gabapentine and buprenorphine) significantly resolved BTZ-induced mechanical allodynia. Results obtained show that CR4056 produces a marked analgesic effect

Bortezomib (BTZ), a selective proteasome inhibitor, is an antitumor drug used to treat multiple myeloma. As a side effect, BTZ determines a painful peripheral neuropathy (PPN), often refractory to management. Chronic BTZ administration in female Wistar rats induced a PPN in which the development of mechanical allodynia is associated to an increase in the expression of TRPV1 and CGRP in the dorsal root ganglia (DRG) and spinal cord dorsal horn. In this study we examine the possible role of two standard analgesics, Gabapentin (Gaba) and buprenorfin (Bupre), in modulating the expression of TRPV1 and CGRP in the DRG and spinal cord of rats chronically treated with BTZ. To this aim, female Wistar rats were treated with BTZ 0.20 mg/kg, 3 times a week for 8 weeks (i.v.). Then Gaba (100 mg/kg, daily, p.o.) and Bupre (28,8 µg/kg, daily, s.c.) were orally administered in a curative schedule for 2 weeks. The expression of TRPV1 and CGRP, a neuropeptide involved in nociceptive neurotransmission, was examined in L4-L5 DRG and spinal cord segments by means of western blot (WB) and immunohistochemistry. WB analysis did not show any statistically significant change of protein levels in DRG and spinal cord. Instead, chronic BTZ treatment followed by a 2 weeks follow-up period affected TRPV1 expression by inducing an increase in the proportion of TRPV1-like immunoreactive (LI) DRG neurons, whereas did not affect the percentage of CGRP-LI neurons. Labeled perikarya were mostly of small- and medium-size. No BTZ-induced changes in immunolabeling were appreciable in the dorsal horn of the spinal cord. Treatment with Gaba and Bupre reduced levels of TRPV1 protein in spinal cord homogenates, whereas had no effect on DRG protein levels. None of the analgesics appeared to reverse in a statistically significant way the BTZ-induced increase of labelled DRG neurons nor induced any change in the detectability of positive innervation in the spinal cord dorsal horn.

Besides the immunomodulatory action, Mesenchymal Stem Cells (MSCs) are able to promote neuronal and glial survival both by releasing trophic factors and through cell to cell contact. For these features MSCs are a promising tool for the treatment of inflammatory and demyelinating diseases such as Multiple Sclerosis (MS). Here we reported a pre-clinical study on Dark Agouti rats affected by a Relapsing-Remitting form of Experimental Autoimmune Encephalomyelitis (RR-EAE), one of the most suitable models for the study of RR-MS. In order to assess the possible preventive or therapeutic effect, 106 MSCs were injected i.v. (intra venous) at day 7 or at day 14 post EAE induction (p.i.) and clinical score was evaluated daily. The preventive schedule of treatment (day 7 p.i.) had no effect on EAE clinical course but the therapeutic one (day 14 p.i.) was able to hamper the relapsing phase from day 19 p.i. and till the end of the experiment (day 45 p.i.) with respect to EAE group. At day 45 p.i., histological analysis performed on spinal cord of EAE rats revealed a substantial absence of inflammatory infiltration and the presence of demyelinated plaques, assessed by Luxol fast Blue staining and by immunohistochemistry for MBP (Myelin Basic Protein). Moreover the analysis performed on serial paraffin sections revealed that the therapeutic schedule with MSCs was able to significantly reduce the extension of demyelinated areas in the spinal cord white matter with respect to EAE and EAE+MSCs day 7 p.i. groups, thus confirming clinical score evaluations. These results suggested that MSCs are able to ameliorate the clinical course of EAE animals by reducing the areas of demyelinated lesions. We are now evaluating the possible mechanism of MSCs action by investigating in vitro some putative myelinating and immunomodulating properties of MSCs. This study was granted by MIUR – FIRB Futuro in Ricerca 2008 RBFR08VSVI_001.

In the last 20 years several in vivo rat models of peripheral neuropathy induced by antineoplastic drugs clarified the phatophysiological mechanisms involved in peripheral neurotoxicity. Moreover these models allowed to identify neuroprotection strategies for preventing sensory nerve and dorsal root ganglia (DRG) neurons damages. Only a few cancer cell lines is able to induce the development of cancer in immunocompetent rats, therefore these models do not represent the best way to investigate, at the same time, the antineoplastic activity and the neurotoxic effects of these drugs; by contrast, murine models are widely employed in oncological studies and can be useful for this purpose. During the Ph.D Programme in Neuroscience, the aim of this projecct has been the neurophysiologic, histophatologic and behavioural characterization of 4 murine models of peripheral neuropathy induced by the proteasome inhibitor bortezomib. The neurotoxic side effects of several antineoplastic agents are among the main reason for tretament modification or their limitation as antitumor therapy. The mechanisms underline the onset of peripheral neurophaties are still not clear, even though it is accepted that the neurotoxicity is dose- and drug-dependent. Bortezomib is a proteasome inhibitor and it is used as an efficient chemotherapeutic agent in the tretament of multiple myeloma. In 2003 the Food and Drug Administration (FDA) approved the use of bortezomib for the treatment of advanced multiple myeloma, then in 2008 the drug was promoted as a first-line chemotherapeutic agent for the treatment of myeloma. However patients treated with bortezomib develop a peripheral neuropathy, often combined with neuropathic pain. This project consist of 3 consecutive phases: (A.1) The development of 4 in vivo murine models of bortezomib-induced peripheral neuropathy. Aim: to evaluate the neuropathologic mechanisms involved in bortezomib-induced peripheral neuropathy in two immunocompetent murine strain

Besides the immunomodulatory action, Mesenchymal Stem Cells (MSCs) are able to promote neuronal and glial survival both by releasing trophic factors and through cell to cell contact. For these features MSCs are a promising tool for the treatment of inflammatory and demyelinating diseases such as Multiple Sclerosis (MS). Here we reported a pre-clinical study on Dark Agouti rats affected by a Relapsing-Remitting form of Experimental Autoimmune Encephalomyelitis (RR-EAE), one of the most suitable models for the study of RR-MS. In order to assess the possible preventive or therapeutic effect, 106 MSCs were injected i.v. (intra venous) at day 7 or at day 14 post EAE induction (p.i.) and clinical score was evaluated daily. The preventive schedule of treatment (day 7 p.i.) had no effect on EAE clinical course but the therapeutic one (day 14 p.i.) was able to hamper the relapsing phase from day 19 p.i. and till the end of the experiment (day 45 p.i.) with respect to EAE group. At day 45 p.i., histological analysis performed on spinal cord of EAE rats revealed a substantial absence of inflammatory infiltration and the presence of demyelinated plaques, assessed by Luxol fast Blue staining and by immunohistochemistry for MBP (Myelin Basic Protein). Moreover the analysis performed on serial paraffin sections revealed that the therapeutic schedule with MSCs was able to significantly reduce the extension of demyelinated areas in the spinal cord white matter with respect to EAE and EAE+MSCs day 7 p.i. groups, thus confirming clinical score evaluations. These results suggested that MSCs are able to ameliorate the clinical course of EAE animals by reducing the areas of demyelinated lesions. We are now evaluating the possible mechanism of MSCs action by investigating in vitro some putative myelinating and immunomodulating properties of MSCs. This study was granted by MIUR – FIRB Futuro in Ricerca 2008 RBFR08VSVI_001.

Cisplatin is an anticancer drug employed for the treatment of several solid tumours, including testicular, ovarian, breast and lung cancers. However, treatment side effects remain an unsolved problem. Some of the patients treated with cisplatin, after a cumulative dose of 300 mg/m2, develop a painful peripheral neuropathy (PPN) characterized by distal paresthesias, numbness, sensory ataxia and neuropathic pain. Since a different degree of the PPN severity suggests an individual variation in the drug toxicity response, the understanding of the genomics underlying PPN would lead to new therapeutic targets and to early diagnostic screening. In this study we assessed the severity of cisplatin-induced PPN in six mouse strains using Nerve Conduction Velocities (NCV), Neurometer studies, morphological analysis of DRG, caudal and sciatic nerves and lumbar spinal cord, morphometrical analysis of DRG and behavioral assessment of mechanical allodynia. Cisplatin 4 mg/Kg was intraperitoneally administered twice a week for 4 weeks in CD1, Balb-c, C57BL6, FVB, DBA and AJ mice. Cisplatin induced a significant impairment of caudal and digital NCV in all except FVB and DBA mice. Moreover, cisplatin administration resulted in a significant decrease in the response to mechanical stimulation in Balb-c, C57BL6, CD1 and DBA starting from the first week of treatment while AJ and FVB never develop mechanical allodynia. Neurometer analysis, that allows a quantitative assessment of the functionality of the three major subpopulations of sensory nerve fibers (A-delta, A-beta and C), showed that large myelinated fibers (A-beta) were affected by cisplatin in Balb-c and CD1, small myelinated (A-delta) only in Balb-c while small unmyelinated fibers (C) were unaffected only in FVB and AJ mice. The morphometrical analysis revealed a nucleolar and somatic atrophy of DRG sensory neurons of CD1 but not of FVB-treated mice. Taken together, these results demonstrate that CD1 and FVB mice were the most and

Multiple sclerosis (MS) is a severe chronic disease characterized by the presence of immuno-mediated demyelinating lesions and impairment of axonal transmission, which cause the reduction of nerve conduction velocity and lead to the development of neurological symptoms. Current therapy for MS is based on immunosuppressant agents, but recently Mesenchymal Stem Cells (MSCs) have been proposed as therapeutic treatment for MS, demonstrating a positive effect when administered before disease onset, mainly due to their immunomodulatory properties. Here, we investigate the therapeutic potential of MSCs into an animal model of multiple sclerosis, represented by Dark Agouti rats affected by chronic Relapsing-Remitting experimental autoimmune encephalomyelitis (EAE). In order to assess their putative effectiveness, 106 MSC were intravenously injected in EAE rats before disease onset (7 days after disease induction), to test the “preventive” schedule, or after disease onset (14 days after MSC induction), to test the “therapeutic” schedule with MSCs. Clinical score was assessed daily, and after 45 days rats were sacrificed and histological analysis of spinal cords were performed to evaluate the demyelinating lesions. Clinical score analysis demonstrated that the “preventive” schedule of treatment had no effect on EAE clinical course, while the therapeutic schedule was able to hamper relapsing phase from day 19 and till the end of the experiment (day 45) with respect to EAE group. At day 45, histological analysis performed on spinal cords of EAE rats demonstrated the presence of demyelinated plaques, assessed by Luxol fast Blue staining and by immunohystochemistry for MBP. The same lesions were present in spinal cords of rats treated with the preventive MSC administration. On the contrary the therapeutic schedule with MSCs was able to significantly reduce the extension of demyelinated areas in the spinal cords, thus confirming clinical score evaluations. These results suggested

Bortezomib (BTZ) is an antineoplastic drug that reversibly binds to an active site threonine in the 26S proteasome. The inhibition of protein degradation in the cells leads to the accumulation of signalling molecules, blocking the tumour survival pathways. BTZ is mainly used for the treatment of multiple myeloma, but unfortunately its clinical use have evidenced the onset of a painful peripheral neuropathy (BIPN) that decreased patients’ quality of life. Despite the clinical relevance of BIPN, our knowledge of the molecular mechanism underlying the painful condition is still limited. In recent studies, inflammation has been described as a possible event in the onset of neuropathic pain induced by some chemotherapy drug. To investigate the putative role of immune response in the BTZ-induced painful peripheral neuropathy, we defined a new immune-suppressed murine model based on sub-lethal X-ray irradiation. Adult female BALB/c mice underwent X-ray irradiation, once a week for 4 weeks with a first dose of 350 RAD and 3 subsequent 100 RAD doses (X-ray group), to deplete the immune cells-mediated response. Part of these animals were treated with BTZ 0.8 mg/kg twice a week for 4 weeks (X-ray+BTZ group). To assess the BTZ-induced neurophysiological damage and the onset of neuropathic pain, nerve conduction velocity and mechanical nociceptive threshold were measured. Sciatic nerve, spinal cord and dorsal root ganglia were processed for light microscope observations. Moreover, flow cytometer analysis of CD45-positive cells were performed in tibiae bone marrow (BM) and peripheral blood (PB) to confirm the immune-suppression. After the first X ray irradiation piloerection and hypokinesia were observed, and significant body weight decreased was measured at the end of treatment in X-ray+BTZ-treated animals. The immune-suppression was persistent in the PB until the end of the study, while a partial recovery in BM was observed in the last time-point evaluation. In addition, BTZ in

Multiple Sclerosis (MS) is a crippling chronic disease of the Central Nervous System caused by the presence of self-antibodies which progressively damage axonal myelin sheath, leading to axonal transmission impairment and to the development of neurological symptoms. MS is characterized by a Relapsing-Remitting course, and current therapies rely only on the use of immunosuppressive drugs, which are however unable to reverse disease progression. Encouraging results have been obtained in preclinical studies with the administration of Mesenchymal Stem Cells (MSCs) before disease onset (Zappia et al., 2005). Here, we investigate the therapeutic potential of MSC administration after disease onset into an animal model of MS, represented by Dark Agouti rats affected by chronic Relapsing-Remitting Experimental Autoimmune Encephalomyelitis (EAE) (Cavaletti et al., 2004). 106 MSC were intravenously injected in EAE rats after disease onset. Clinical score was assessed daily, and after 45 days rats were sacrificed and histological analysis of spinal cords performed to evaluate the demyelinating lesions. After the first peak of disease, no further relapses were observed in EAE rats treated with MSCs, differently from what observed in EAE group. Histological analysis demonstrated the presence of demyelinated plaques in spinal cords of EAE rats, (Luxol fast Blue staining and anti-MBP immunohystochemistry). On the contrary the therapeutic schedule with MSCs significantly reduces the number and the extension of demyelinated areas in the spinal cords, confirming clinical score evaluations. These results demonstrated that MSCs ameliorate the clinical course of EAE and hamper the disease relapsing by reducing the areas of demyelinated lesions.

Enterococcus faecalis isolates from food and environmental origin were evaluated for their angiotensin-converting enzyme (ACE)-inhibitory activity (ACE-IA) after growth in bovine skim milk (BSM). Most (90% active) but not all (10% inactive) E. faecalis strains produced BSM-derived hydrolysates with high ACE-IA. Known ACE-inhibitory peptides (ACE-IP) and an antioxidant peptide were identified in the E. faecalis hydrolysates by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (RP-HPLC-MS/MS). Antimicrobial activity against Pediococcus damnosus CECT4797 and Listeria ivanovii CECT913 was also observed in the E. faecalis hydrolysates. The incidence of virulence factors in the E. faecalis strains with ACE-IA and producers of ACE-IP was variable but less virulence factors were observed in the food and environmental strains than in the clinical reference strains. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) based analysis demonstrated that food and environmental E. faecalis strains were genetically different from those of clinical origin. When evaluated, most E. faecalis strains of clinical origin also originated BSM-derived hydrolysates with high ACE-IA due to the production of ACE-IP. Accordingly, the results of this work suggest that most E. faecalis strains of food, environmental and clinical origin produce BSM-derived bioactive peptides with human health connotations and potential biotechnological applications. © 2013 Elsevier B.V.

Bortezomib (BTZ) is an effective antineoplastic drug for the treatment of multiple myeloma. Its clinical use induce the development of a peripheral neuropathy characterized by sensory alterations and neuropathic pain. Rat models of BTZ-induced peripheral neuropathy had been established. However, since only a few cancer cell lines induce the development of cancer in the rat, these models don’t represent the most effective way to study, at the same time, the antineoplastic activity and the neurotoxic effects of BTZ. Here, immunodeficient SCID mice were s.c injected with RPMI8266 human myeloma cells. Three weeks after tumour injection, mice were i.v treated with BTZ 1 mg/kg once a week for five weeks. The tumour volume was assessed as a measure of BTZ antitumor activity; the mechanical nociceptive threshold and nerve conduction velocities were measured to assess the neuropathic pain and the toxic effect of BTZ on the peripheral nervous system, respectively. Starting from the first administration, BTZ 1 mg/Kg was able to block tumour-growth but inducing the development of mechanical allodynia and an impairment of nerve functions. Interestingly, myeloma itself seemed to be able to induce mild functional alterations of peripheral nerves. This mouse model and treatment schedule allowed the study of the antineoplastic activity and of the neurotoxic effects of BTZ at the same time. Moreover, a preliminary evaluation of the effect of myeloma itself on the peripheral nervous system was assessed. This animal model should be used in the preclinical discovery of new neuroprotective as well as of analgesic compounds. This work was partially sponsored by POR della Lombardia OB. 2 FSE 2007-2013, Asse IV - Sviluppo del Capitale Umano

Bortezomib is an antineoplastic agents that is often used to treat the multiple myeloma and of some lymphomas. Although its well-known antitumor activity, its effectiveness is limited by the highly incidental development of severe peripheral neuropathy (BIPN). This neuropathy is characterized by dysesthesia, numbness and a painful sensations. In order to obtain a pre-clinical model to improve our understanding of BIPN molecular pathways, we performed an rat model in which bortezomib (0,20 mg/kg) was administered three times weekly for eight weeks, followed by a four-week follow up period. At the end of the treatment period, we assessed nerve conduction velocity (NCV) and pathological changes in caudal nerve, dorsal root ganglia (DRGs), and sciatic nerve. Afterwards, we verified the involvement of proteasome inhibition and also we evaluated the microtubule stability in sciatic nerve by comparing the distribution of acetylated tubulin between polymerized (P) and soluble (S) fractions by western blot experiments. The neurophysiological evaluation demonstrated a reduction in NCV both at eight weeks and after the follow up period: at the pathologic analysis, caudal nerves were the most affected structures, whereas DRGs showed a vacuolization in the cytoplasm in sensory neurons and of satellite cells, although the sciatic nerves evidenced a mild axonopathy. Proteasome activity assay was performed on peripheral blood mononuclear cells (PBMCs), sciatic nerve and brain: PBMCs and sciatic nerve recovered quickly in the acute setting, while maintaining strong inhibition until 48 hours after last injection when the drug was chronically administered. Moreover, at six and eight weeks of treatment, we observed the increase of acetylated alpha-tubulin in the polymerized fraction in sciatic nerves of BTZ-treated animals as compared with control, that returning at baseline during follow up period. In conclusion, our results demonstrated that BTZ is able to induce a toxic effect on pe

Cisplatin (CDDP), Taxol (TAX) and Bortezomib (BZ) are chemotherapeutic drugs commonly employed in clinical practice for the treatment of solid and haematological tumors. Their clinical use is limited by the development of a peripheral neuropathy characterized by sensory alterations, pain and, in part, by motor and autonomic dysfunctions. Several rat models had been performed to study and describe the mechanisms of the peripheral neurotoxicity induced by these drugs. However, since only few cancer cell lines are able to induce the development of cancer in the rat, we focused our attention on mice models to allow the combined study of the antineoplastic activity and of the neurotoxic effects of the anticancer compounds. Moreover, different mice strains should be useful for different kind of studies. Here we investigated the interactions between the mice genotype and their drug response to determine the susceptibility to the development of chemotherapy-induced peripheral neuropathy. To this aim, we investigated the neurophysiological and neuropathological alterations induced by the chronic treatment with different chemotherapy drugs in female Balb/c, CD1 and c57 mice and in male c57 and FVB mice. Mice were injected with Cisplatin (2, 4 mg/Kg, ip, 2qw), or Taxol (50, 70, 80 mg/Kg, iv, 1qw) or Bortezomib (0.4, 0.8 mg/Kg, iv, 2qw) for a period of 4-6 weeks. At the end of the treatment the nerve conduction velocities (NCVs) were determined in the caudal and in the digital nerves and the dorsal root ganglia (DRG) and sciatic nerves collected for the neuropathological analysis; behavioural tests were performed to study if these drugs are able to induce allodynia or hyperalgesia. In all mice models we observed that TAX and CDDP induced a significant alteration in body weight at the end of the treatment. All the drugs induced a significant reduction in the caudal and digital NCV in Balb/c and CD1 mice. TAX and BZ caused the axonal degeneration of the sciatic nerves while CDDP

Objective: Neutrophils have been shown to own several immunoregulatory properties. Nevertheless few attention has been given to their contribution to both Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis (EAE) etiopathogenesis and progression. We aim to investigate the potential role of neutrophils during EAE sensitization and chronic phase in Dark Agouti (DA) EAE. Methods: Chronic EAE was induced in DA rats by intrafootpad injection of syngenic spinal cord homogenate in incomplete Freund's adjuvant. Neutrophils were depleted by means of intraperitoneal injection of anti-rat neutrophil serum (aNEU) at different disease time course, i.e. either during the sensitization and the chronic phase of the disease. The animals were weighted weekly and aNEU effect was first evaluated on clinical score while deeper analysis were performed on blood formula, spleen morphology and spinal cord cytokine, chemokine and myelin basic protein expression. Results: aNEU had no effect on clinical score when neutrophil depletion was performed during the chronic phase, i.e. from 14 to 35 days post EAE induction (dpi). When aNEU was administered during the EAE sensitization phase up to 8dpi, no difference in mean clinical score could be revealed between EAE and aNEU-treated EAE rats up to the disease peak, i.e. 14 dpi. However a significant improvement in clinical condition could be reported soon after the disease peak so that a chronic course was transformed into an acute/ monophasic one. aNEU treatment induced a faster body weight gain while it did not affect blood formula changes related to EAE course. Moreover while aNEU treatment did not counteract EAE effects on both spleen microscopic morphological changes and Treg (FoxP3) content, it mainly affected the expression of both pro- and anti-inflammatory cytokines in the three main spinal cord traits (cervical, thoracic, lumbar) while relative low effect was observed on both MCP-1 and myelin basic protein expression. Conclus

Bortezomib (BTZ) is a chemotherapy drug with clinical efficacy in patients with relapsed/refractory, relapsed and newly diagnosed multiple mieloma (MM). It acts by inhibiting protein degradation by the proteasoma, but its clinical use is limited by a dose-limiting neurotoxicy and by the development of tumor resistance. BTZ-induced peripheral neuropathy may occur in cancer patients from a variety of mechanisms, among which proteasoma inhibition and the alteration of microtubule’s stability. To study in vivo these two different mechanisms involved in the onset of neuropathy, we used a well-characterized rat model of BTZ-induced peripheral neuropathy. Wistar rat were administered with BTZ 0.20 mg/kg, three times/week for eight weeks. At the end of the treatment period the relationship between neurotoxicity and the profile of the proteasoma inhibition was evaluated in different tissues. The level of 20S proteasoma inhibition was assessed by the proteasoma activity assay into blood mononuclear cells (PBMC), sciatic nerve and brain by fluorimetric assay at different time points. Moreover, we examined microtubule polymerization in sciatic nerve by comparing the distribution of acetylated tubulin (a post translational marker of stabilized microtubule) between polymerized (P) and soluble (S) fractions by western blot experiments. After eight weeks of treatment, we observed BTZ-related neurophysiologic alterations and neuropathological damages in the axons of sciatic nerves and in sensory neurons and satellite cells of dorsal root ganglia, demonstrating the onset of a peripheral neuropathy. The recovery of the proteasoma activity was observed within 24 hours from the drug administration if BTZ was injected in a single acute dose; while the proteasoma activity remained suppressed if the drug was chronically administered. This effect was probably due to a cumulative effect of chronic administration of BTZ on its biological target. Besides, in this study we increase in the amoun

The clinical use of cisplatin (CDDP) is often associated with a severe neurotoxicity that represents the dose-limiting factor in therapy. In the past, several rat models has been accomplished to study the mechanisms involved in peripheral neuropathy induced by chronic CDDP administration and they highlight its toxic effects particularly in the dorsal root ganglia (DRG) sensory neurons that are smaller in sizes. Nevertheless, since only a limited number of cancer cell lines are able to induce the development of cancer in rat, we decided to use different murine models that allow a future combined study of the antineoplastic activity and the neurotoxic effects of CDDP. To achieve this aim, we evaluated the neurophysiological, neuropathological and morphometrical alterations induced by chronic CDDP treatment in female Balb/c, CD1 and Hola Hsd NuNu mice. Balb/c mice were treated intraperitoneally with CDDP 4mg/Kg twice a week for 4 weeks, while CD1 and Hola Hsd NuNu mice were treated with the same schedule followed by 2 weeks of follow-up. The body weight was measured once a week while at the end of the treatment we evaluated the nerve conduction velocities (NCVs) in the caudal and digital nerves and we collected DRG for the morphological and morphometrical analysis. We observed in all mice models a significant decrease in the body weight and a significant reduction in the caudal and digital NCV. This nerve functional damage was confirmed by morphological observations in DRG showing an increase in multinucleolated neurons and in nucleolar eccentricity and by the presence of nucleolar segregation. Moreover the morphometrical analysis demonstrated that CDDP affects neurons through a significant decrease in the size of somatic and nucleolar area in Balb/c mice and also in nuclear size in CD1 mice while the Nu/Nu mice showed only a significant decrease in the nuclear area. These results can confirm that the chosen schedule is able to induce peripheral neuropathy at rather di

Since peripheral sensory neuropathy is the major, clinically relevant side effect of sagopilone we investigated the general and peripheral neurotoxicity of sagopilone administered intravenously with different doses (1.2 and 2.4 mg/kg) and schedules in 48 Wistar rats and we performed in parallel a pharmacokinetic/pharmacodynamic (PK/PD) study. A trend toward a different peripheral neurotoxicity could be assessed after 2 weeks of treatment (bolus > 30-min infusion > 3-h infusion) with both doses of sagopilone. Although sagopilone concentrations in peripheral nerve tissue above 100 ng/g were associated with a reduction in nerve conduction velocity (NCV), a clear dose-dependence of this reduction on the level of systemic exposure to sagopilone was not observed. The PK/PD evaluation revealed no consistent effect of the infusion duration on serum PK parameters or the PD read-out NCV. Sagopilone concentrations in brain, sciatic nerve, liver, and kidney were higher after bolus compared to infusion, but there were no influence of infusion duration on these concentrations. No correlation between sagopilone concentrations in any organ/tissue with NCV changes was detected. This study evidences that the PD of sagopilone is not the main determinant of the onset and severity of sagopilone-induced peripheral neurotoxicity in the investigated clinically-relevant dose range, thus indicating that further investigation might identify neuronal-specific mechanisms of action able to drive a focused strategy to prevent peripheral neurotoxicity without reducing the anticancer effectiveness of the epothilones.

Bortezomib (BTZ) is an effective antineoplastic drug for the treatment of multiple myeloma. Its clinical use induce the development of a peripheral neuropathy characterized by sensory alterations and neuropathic pain. Rat models of BTZ-induced peripheral neuropathy had been established. However, since only a few cancer cell lines induce the development of cancer in the rat, these models don’t represent the most effective way to study, at the same time, the antineoplastic activity and the neurotoxic effects of BTZ. Here, immunodeficient SCID mice were s.c injected with RPMI8266 human myeloma cells. Three weeks after tumour injection, mice were i.v treated with BTZ 1 mg/kg once a week for five weeks. The tumour volume was assessed as a measure of BTZ antitumor activity; the mechanical nociceptive threshold and nerve conduction velocities were measured to assess the neuropathic pain and the toxic effect of BTZ on the peripheral nervous system, respectively. Starting from the first administration, BTZ 1 mg/Kg was able to block tumour-growth but inducing the development of mechanical allodynia and an impairment of nerve functions. Interestingly, myeloma itself seemed to be able to induce mild functional alterations of peripheral nerves. This mouse model and treatment schedule allowed the study of the antineoplastic activity and of the neurotoxic effects of BTZ at the same time. Moreover, a preliminary evaluation of the effect of myeloma itself on the peripheral nervous system was assessed. This animal model should be used in the preclinical discovery of new neuroprotective as well as of analgesic compounds. This work was partially sponsored by POR della Lombardia OB. 2 FSE 2007-2013, Asse IV - Sviluppo del Capitale Umano

Cisplatin (CDDP), Taxol (TAX) and Bortezomib (BZ) are chemotherapeutic drugs commonly employed in clinical practice for the treatment of solid and haematological tumors. Their clinical use is limited by the development of a peripheral neuropathy characterized by sensory alterations, pain and, in part, by motor and autonomic dysfunctions. Several rat models had been performed to study and describe the mechanisms of the peripheral neurotoxicity induced by these drugs. However, since only few cancer cell lines are able to induce the development of cancer in the rat, we focused our attention on mice models to allow the combined study of the antineoplastic activity and of the neurotoxic effects of the anticancer compounds. Moreover, different mice strains should be useful for different kind of studies. Here we investigated the interactions between the mice genotype and their drug response to determine the susceptibility to the development of chemotherapy-induced peripheral neuropathy. To this aim, we investigated the neurophysiological and neuropathological alterations induced by the chronic treatment with different chemotherapy drugs in female Balb/c, CD1 and c57 mice and in male c57 and FVB mice. Mice were injected with Cisplatin (2, 4 mg/Kg, ip, 2qw), or Taxol (50, 70, 80 mg/Kg, iv, 1qw) or Bortezomib (0.4, 0.8 mg/Kg, iv, 2qw) for a period of 4-6 weeks. At the end of the treatment the nerve conduction velocities (NCVs) were determined in the caudal and in the digital nerves and the dorsal root ganglia (DRG) and sciatic nerves collected for the neuropathological analysis; behavioural tests were performed to study if these drugs are able to induce allodynia or hyperalgesia. In all mice models we observed that TAX and CDDP induced a significant alteration in body weight at the end of the treatment. All the drugs induced a significant reduction in the caudal and digital NCV in Balb/c and CD1 mice. TAX and BZ caused the axonal degeneration of the sciatic nerves while CDDP

Bortezomib (BTZ) is a chemotherapy drug with clinical efficacy in patients with relapsed/refractory, relapsed and newly diagnosed multiple mieloma (MM). It acts by inhibiting protein degradation by the proteasoma, but its clinical use is limited by a dose-limiting neurotoxicy and by the development of tumor resistance. BTZ-induced peripheral neuropathy may occur in cancer patients from a variety of mechanisms, among which proteasoma inhibition and the alteration of microtubule’s stability. To study in vivo these two different mechanisms involved in the onset of neuropathy, we used a well-characterized rat model of BTZ-induced peripheral neuropathy. Wistar rat were administered with BTZ 0.20 mg/kg, three times/week for eight weeks. At the end of the treatment period the relationship between neurotoxicity and the profile of the proteasoma inhibition was evaluated in different tissues. The level of 20S proteasoma inhibition was assessed by the proteasoma activity assay into blood mononuclear cells (PBMC), sciatic nerve and brain by fluorimetric assay at different time points. Moreover, we examined microtubule polymerization in sciatic nerve by comparing the distribution of acetylated tubulin (a post translational marker of stabilized microtubule) between polymerized (P) and soluble (S) fractions by western blot experiments. After eight weeks of treatment, we observed BTZ-related neurophysiologic alterations and neuropathological damages in the axons of sciatic nerves and in sensory neurons and satellite cells of dorsal root ganglia, demonstrating the onset of a peripheral neuropathy. The recovery of the proteasoma activity was observed within 24 hours from the drug administration if BTZ was injected in a single acute dose; while the proteasoma activity remained suppressed if the drug was chronically administered. This effect was probably due to a cumulative effect of chronic administration of BTZ on its biological target. Besides, in this study we increase in the amoun

Bortezomib is an antineoplastic agents that is often used to treat the multiple myeloma and of some lymphomas. Although its well-known antitumor activity, its effectiveness is limited by the highly incidental development of severe peripheral neuropathy (BIPN). This neuropathy is characterized by dysesthesia, numbness and a painful sensations. In order to obtain a pre-clinical model to improve our understanding of BIPN molecular pathways, we performed an rat model in which bortezomib (0,20 mg/kg) was administered three times weekly for eight weeks, followed by a four-week follow up period. At the end of the treatment period, we assessed nerve conduction velocity (NCV) and pathological changes in caudal nerve, dorsal root ganglia (DRGs), and sciatic nerve. Afterwards, we verified the involvement of proteasome inhibition and also we evaluated the microtubule stability in sciatic nerve by comparing the distribution of acetylated tubulin between polymerized (P) and soluble (S) fractions by western blot experiments. The neurophysiological evaluation demonstrated a reduction in NCV both at eight weeks and after the follow up period: at the pathologic analysis, caudal nerves were the most affected structures, whereas DRGs showed a vacuolization in the cytoplasm in sensory neurons and of satellite cells, although the sciatic nerves evidenced a mild axonopathy. Proteasome activity assay was performed on peripheral blood mononuclear cells (PBMCs), sciatic nerve and brain: PBMCs and sciatic nerve recovered quickly in the acute setting, while maintaining strong inhibition until 48 hours after last injection when the drug was chronically administered. Moreover, at six and eight weeks of treatment, we observed the increase of acetylated alpha-tubulin in the polymerized fraction in sciatic nerves of BTZ-treated animals as compared with control, that returning at baseline during follow up period. In conclusion, our results demonstrated that BTZ is able to induce a toxic effect on pe

The clinical use of cisplatin (CDDP) is often associated with a severe neurotoxicity that represents the dose-limiting factor in therapy. In the past, several rat models has been accomplished to study the mechanisms involved in peripheral neuropathy induced by chronic CDDP administration and they highlight its toxic effects particularly in the dorsal root ganglia (DRG) sensory neurons that are smaller in sizes. Nevertheless, since only a limited number of cancer cell lines are able to induce the development of cancer in rat, we decided to use different murine models that allow a future combined study of the antineoplastic activity and the neurotoxic effects of CDDP. To achieve this aim, we evaluated the neurophysiological, neuropathological and morphometrical alterations induced by chronic CDDP treatment in female Balb/c, CD1 and Hola Hsd NuNu mice. Balb/c mice were treated intraperitoneally with CDDP 4mg/Kg twice a week for 4 weeks, while CD1 and Hola Hsd NuNu mice were treated with the same schedule followed by 2 weeks of follow-up. The body weight was measured once a week while at the end of the treatment we evaluated the nerve conduction velocities (NCVs) in the caudal and digital nerves and we collected DRG for the morphological and morphometrical analysis. We observed in all mice models a significant decrease in the body weight and a significant reduction in the caudal and digital NCV. This nerve functional damage was confirmed by morphological observations in DRG showing an increase in multinucleolated neurons and in nucleolar eccentricity and by the presence of nucleolar segregation. Moreover the morphometrical analysis demonstrated that CDDP affects neurons through a significant decrease in the size of somatic and nucleolar area in Balb/c mice and also in nuclear size in CD1 mice while the Nu/Nu mice showed only a significant decrease in the nuclear area. These results can confirm that the chosen schedule is able to induce peripheral neuropathy at rather di

Objective: Neutrophils have been shown to own several immunoregulatory properties. Nevertheless few attention has been given to their contribution to both Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis (EAE) etiopathogenesis and progression. We aim to investigate the potential role of neutrophils during EAE sensitization and chronic phase in Dark Agouti (DA) EAE. Methods: Chronic EAE was induced in DA rats by intrafootpad injection of syngenic spinal cord homogenate in incomplete Freund's adjuvant. Neutrophils were depleted by means of intraperitoneal injection of anti-rat neutrophil serum (aNEU) at different disease time course, i.e. either during the sensitization and the chronic phase of the disease. The animals were weighted weekly and aNEU effect was first evaluated on clinical score while deeper analysis were performed on blood formula, spleen morphology and spinal cord cytokine, chemokine and myelin basic protein expression. Results: aNEU had no effect on clinical score when neutrophil depletion was performed during the chronic phase, i.e. from 14 to 35 days post EAE induction (dpi). When aNEU was administered during the EAE sensitization phase up to 8dpi, no difference in mean clinical score could be revealed between EAE and aNEU-treated EAE rats up to the disease peak, i.e. 14 dpi. However a significant improvement in clinical condition could be reported soon after the disease peak so that a chronic course was transformed into an acute/ monophasic one. aNEU treatment induced a faster body weight gain while it did not affect blood formula changes related to EAE course. Moreover while aNEU treatment did not counteract EAE effects on both spleen microscopic morphological changes and Treg (FoxP3) content, it mainly affected the expression of both pro- and anti-inflammatory cytokines in the three main spinal cord traits (cervical, thoracic, lumbar) while relative low effect was observed on both MCP-1 and myelin basic protein expression. Conclus

Cisplatin (CDDP) and Bortezomib (BTZ) are two antineoplastic drugs commonly used against different solid tumors and multiple myeloma, respectively. Although their efficacy, the clinical employment of these chemoterapeutics is associated with a severe neurotoxicity that represents the dose-limiting factor in therapy. Previous well established rat models evidenced the toxic effects of CDDP particularly on the dorsal root ganglia (DRG) sensory neurons that are smaller in the somatic, nuclear and nucleolar sizes. To investigate the role of CDDP and BTZ on the peripheral nervous system we used three mice models: female Balb/c were treated with CDDP 4mg/Kg (ip, 2qwx4) and with BTZ 0,8 mg/Kg (iv, 2qwx4), while female Hola Hsd NuNu and CD1 mice were treated with CDDP 4mg/Kg (ip 2qwx4 followed by 2 weeks of follow-up) and with BTZ 0,8 mg/Kg (iv, 2qwx6). The aim of this study was to perform a morphometrical quantification of cisplatin and bortezomib-induced toxic effects on the DRG neurons in the different mice strains. Three L4-L5 DRG/animals were collected, fixed and resin-embedded; 1 micro-sections stained with toluidine blu were analyzed with a computer-assisted image analyzer (Image J NIH sotware). In randomly selected sections the somatic, nuclear and nucleolar sizes of DRG sensory neurons were measured on at least 200 DRG neurons/animal. The morphometrical analysis of DRG showed that the administration of CDDP affects neurons causing a decrease in the size of somatic and nucleolar area in the Balb/c and CD1mice while the Nu/Nu strain showed only a decrease in the nuclear area. By contrast, the administration of BTZ induced an increase in the size of somatic, nuclear and nucleolar area in the Balb/c mice while only nucleolar area was increased in Nu/Nu and CD1 mice. The results obtained with the administration of CDDP seems to confirm the previous studies in rat, showing the toxic role of CDDP on neurons of DRG. Since the BTZ-induced increase in the cellular sizes seems