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Chemotherapy-induced painful peripheral neurotoxicity (painful-CIPN), with paresthesia, numbness, dysaesthesia, and neuropathic pain ranks among the most common dose-limiting toxicity of widely used anticancer drugs. Beside peripheral neurons, for several years considered the only reasonable target for painful-CIPN study, the recent evaluation of the microvascular angiogenesis in the somatosensory pathway, reveals other important actors in the neuropathic pain development and chronicization. To elucidate the relation between chemotherapy-induced neuropathic pain and vascular alterations, we evaluated the microvasculature in central and peripheral nervous compartments of rats exposed to neurotoxic chemotherapy. Rats were treated with paclitaxel 10 mg/kg once a week for 4 weeks, or with cisplatin 2mg/kg twice a week for 4 weeks or with their vehicles. Animals were tested for neurophysiological abnormalities and pain before and after the treatments. Post-mortem samples were analyzed at synchrotron radiation sources by X-ray Phase-Contrast Tomography (XPCT) Imaging and processed for quantitative and morphological analyses of microvascular structures. Complementarily, histochemical and molecular evaluations were performed to validate the results. XPCT analysis revealed that rats exposed to paclitaxel (affected by a painful sensory axonopathy) showed an increased vascular density (putative sprouting angiogenesis) in the crucial districts of the central (somatosensory cortex and lumbar spinal cord) and peripheral nervous system (lumbar Dorsal Root Ganglia and peripheral nerves). However, the complexity of the vascular network and the size of neo-formed vessels were significantly decreased in some specific regions. On the other hand, no significant changes were observed in rats exposed to CDDP (affected by a painless mild neuronopathy) suggesting a specific involvement of neo-angiogenesis in the development of neuropathic pain. Molecular analysis performed on the DRG and S1

Paclitaxel-induced peripheral neurotoxicity (PIPN) is a potentially dose-limiting side effect in anticancer chemotherapy. Several animal models of PIPN exist, but their results are sometimes difficult to be translated into the clinical setting. We compared 2 widely used PIPN models characterized by marked differences in their methodologies. Female C57BL/6JOlaHsd mice were used, and they received only paclitaxel vehicle (n = 38) or paclitaxel via intravenous injection (n = 19, 70 mg/kg) once a week for 4 weeks (Study 1) or intraperitoneally (n = 19, 10 mg/kg) every 2 days for 7 times (Study 2). At the end of treatment and in the follow-up, mice underwent behavioral and neurophysiological assessments of PIPN. At the same time points, some mice were killed and dorsal root ganglia, skin, and sciatic and caudal nerve samples underwent pathological examination. Serum neurofilament light levels were also measured. The differences in the neurotoxicity parameters were analyzed using a nonparametric Mann-Whitney test, with significance level set at P < 0.05. Study 1 showed significant and consistent behavioral, neurophysiological, pathological, and serological changes induced by paclitaxel administration at the end of treatment, and most of these changes were still evident in the follow-up period. By contrast, study 2 evidenced only a transient small fiber neuropathy, associated with neuropathic pain. Our comparative study clearly distinguished a PIPN model recapitulating all the clinical features of the human condition and a model showing only small fiber neuropathy with neuropathic pain induced by paclitaxel.

The proteasomal system is involved in the turnover of damaged proteins and it is responsible for their degradation. Because of its role in oncogenesis, the inhibition of the proteasome system is a promising therapeutic target for neoplastic treatment. The accumulation and deleterious effects of toxic proteins are frequently induced by exposure to chemotherapeutic drugs and 20S proteasome inhibitors, such as bortezomib (BTZ) and carfilzomib (CFZ) have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of multiple myeloma (MM) and some other liquid tumours. Although the survival of MM patients has been improved by the introduction of both drugs-based therapies, these proteasome inhibitors have several limitations, including chemotherapy-induced peripheral neurotoxicity (CIPN). Since it is becoming increasingly clear that cytoskeletal integrity and mitochondrial function are intricately linked, we compared these targets after BTZ and CFZ treatments in vitro. Primary culture of dorsal root ganglion (DRG) sensory neurons isolated from adult mice were treated with BTZ 10 nM and CFZ 60 nM for 24 hours, and measurement of energy metabolism were investigated using XFe24 Seahorse Analyzer, while a morphological analysis of mitochondria was performed in silico using the toolset MiNA (Mitochondrial Network Analysis). Moreover, by immunoblotting we have evaluated drug-induced alterations of proteins involved in cytoskeleton, mitochondrial oxidative phosphorylation, and molecular motors transport. BTZ was shown to induce several cytoskeletal damage in terms of increased levels of delta2-tubulin, acetylated tubulin and MAP2 expressions compared to both CFZ-treated cells and untreated cells. Conversely, the evaluation of the bioenergetic mitochondrial metabolism revealed a reduction in basal and maximal respiration for both chemotherapeutic drugs. Similarly, both BTZ-and CFZ cultures showed a decrease in ATP production compared with the untreated cells.

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.

Multicentre preclinical randomized controlled trials (pRCTs) are a valuable tool to improve experimental stroke research, but are challenging and therefore underused. A common challenge regards the standardization of procedures across centres. We here present the harmonization phase for the quantification of sensorimotor deficits by composite neuroscore, which was the primary outcome of two multicentre pRCTs assessing remote ischemic conditioning in rodent models of ischemic stroke. Ischemic stroke was induced by middle cerebral artery occlusion for 30, 45 or 60 min in mice and 50, 75 or 100 min in rats, allowing sufficient variability. Eleven animals per species were video recorded during neurobehavioural tasks and evaluated with neuroscore by eight independent raters, remotely and blindly. We aimed at reaching an intraclass correlation coefficient (ICC) ≥0.60 as satisfactory interrater agreement. After a first remote training we obtained ICC = 0.50 for mice and ICC = 0.49 for rats. Errors were identified in animal handling and test execution. After a second remote training, we reached the target interrater agreement for mice (ICC = 0.64) and rats (ICC = 0.69). In conclusion, a multi-step, online harmonization phase proved to be feasible, easy to implement and highly effective to align each centre’s behavioral evaluations before project’s interventional phase.

Chemotherapy-induced peripheral neuropathy (CIPN) is a frequent side effect of various cancer chemotherapy treatments. Typically, CIPN manifests in patients as numbness, tingling, altered sensation, usually associated with neuropathic pain and, therefore the adjustment of chemotherapy dosages or even discontinuation of the treatment. Although various chemotherapeutic agents induce neuropathy, they do so by targeting different cellular processes and having different mechanisms. The pathomechanism by which chemotherapeutics damage the nervous is multifactorial and involves microtubule disruption, oxidative stress and mitochondrial damage, altered ion channel activity, myelin sheath damage, DNA damage, immunological processes and neuroinflammation. Neuroinflammation is one of the host defensive mechanisms and occurs as one of the most common pathological outcomes in most neurological and neurodegenerative diseases make it the promising target. Several CIPN preclinical studies suggest critical neuro-immune interactions as a mechanism of neurotoxicity. In this study we analyzed the presence of neuroinflammatory marker proteins in rats treated with chemotherapy drugs, in order to identify the neuroinflammasome as a pathological mechanism. Expression of inflammasome protein NLRP3, interleukin IL6 and chemokine CCL2 was analyzed in nervous tissue (DRG, spinal cord, caudal nerve, sciatic nerve) after chemotherapy with Paclitaxel, Oxaliplatin and Bortezomib. All three proteins showed a significant increase in the caudal nerve in Paclitaxel treatment over 4 weeks. Treatment with Oxaliplatin did not give any increase in protein expression, while Bortezomib showed a significant increase only for CCL2. Immunohistochemical staining represents infiltration of macrophages in caudale nerves in rats treated with Paclitaxel and Borthezomib but not Oxaliplatin. Neurophysiology and dynamic tests showed that rats treated with Paclitaxel and Bortezomib develop allodynia, reduce nerve condu

Background and Aims: Chemotherapy-induced peripheral neurotoxicity (CIPN) is a common and long-lasting adverse event of several anticancer compounds, for which treatment has not yet been developed. To fill this gap, preclinical studies are warranted, exploiting highly translational outcome measure(s) to transfer data from bench to bedside. Nerve excitability testing (NET) enables to test in vivo axonal properties and can be used to monitor early changes leading to axonal damage. Methods: We tested NET use in two different CIPN rat models: oxaliplatin (OHP) and paclitaxel (PTX). Animals (female) were chronically treated with either PTX or OHP and compared to respective control animals. NET was performed as soon as the first injection was administered. At the end of the treatment, CIPN onset was verified via a multimodal and robust approach: nerve conduction studies, nerve morphometry, behavioural tests and intraepidermal nerve fibre density. Results: NET showed the typical pattern of axonal hyperexcitability in the 72 h following the first OHP administration, whereas it showed precocious signs of axonal damage in PTX animals. At the end of the month of treatment, OHP animals showed a pattern compatible with a mild axonal sensory polyneuropathy. Instead, PTX cohort was characterised by a rather severe sensory axonal polyneuropathy with minor signs of motor involvement. Interpretation: NET after the first administration demonstrated the ongoing OHP-related channelopathy, whereas in PTX cohort it showed precocious signs of axonal damage. Therefore, NET could be suggested as an early surrogate marker in clinical trials, to detect precocious changes leading to axonal damage.

Oxaliplatin (OHP) chemotherapy (CHT) is seriously limited by neurotoxic side effects for whom there is no treatment; this unmet clinical need is in part due to an uncompleted pathogenetic knowledge and, therefore, robust preclinical models are needed to advance patients’ care. OHP-induced peripheral neurotoxicity (OIPN) has a peculiar profile: it comprises an acute syndrome and a chronic sensory axonopathy. Acute OIPN is characterized by transient cold-induced paresthesia and cramps, lasting 2-3 days after each administration; acute OIPN has been attributed to a transient ion channel dysfunction. The worse acute OIPN is, the more severe the chronic neuropathy that ensues. Therefore, an OIPN model should be able to reproduce both conditions. We designed an in vivo study to this aim: we compared a control group with a treated group (OHP 3 mg/Kg twice a week over 4 weeks, iv). Nerve excitability testing (NET) was used to assess acute OIPN. Behavioural test, nerve conduction studies (NCS), and neuropathology were used to characterise chronic OIPN; the latter included: morphological/morphometrical assessments of the caudal nerve and dorsal root ganglia (DRG); intraepidermal nerve fiber density (IENFD); spinal cord immunohistochemistry for the transient receptor potential vanilloid type-1 (TRPV1) receptor. Data were collected at the end of treatment and 6 weeks after. NET allowed us to show that acute OIPN ensued as soon as the first administration and it did not persist after CHT (no NET alterations 1 week after CHT completion). Behavioural tests, NCS, nerve/DRG morphological/morphometrical analysis, and IENFD showed that a mild sensory neuronopathy/axonopathy had ensued at CHT completion and nearly completely resolved at follow-up. Densitometric analysis of TRPV1 immunolabeling in the dorsal horn of the spinal cord at the end of treatment showed an increased density of TRPV1 staining in OHP animals (in lamina I and inner lamina II). This difference was maintained at fol

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.

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.

Dorsal root ganglia (DRGs) are clusters of sensory neurons that transmit the sensory information from the periphery to the central nervous system, and satellite glial cells (SGCs), their supporting trophic cells. Sensory neurons are pseudounipolar neurons with a heterogeneous neurochemistry reflecting their functional features. DRGs, not protected by the blood brain barrier, are vulnerable to stress and damage of different origin (i.e., toxic, mechanical, metabolic, genetic) that can involve sensory neurons, SGCs or, considering their intimate intercommunication, both cell populations. DRG damage, primary or secondary to nerve damage, produces a sensory peripheral neuropathy, characterized by neurophysiological abnormalities, numbness, paraesthesia and dysesthesia, tingling and burning sensations and neuropathic pain. DRG stress can be morphologically detected by light and electron microscope analysis with alterations in cell size (swelling/atrophy) and in different sub-cellular compartments (i.e., mitochondria, endoplasmic reticulum, and nucleus) of neurons and/or SGCs. In addition, neurochemical changes can be used to portray abnormalities of neurons and SGC. Conventional immunostaining, i.e., immunohistochemical detection of specific molecules in tissue slices, can be employed to detect, localize and quantify particular markers of damage in neurons (i.e., nuclear expression of ATF3) or SGCs (i.e., increased expression of GFAP), markers of apoptosis (i.e., caspases), markers of mitochondrial suffering and oxidative stress (i.e., 8-OHdG), markers of tissue inflammation (i.e., CD68 for macrophage infiltration) etc. However classical (2D) methods of immunostaining disrupt the overall organization of the DRG, thus resulting in the loss of some crucial information. Whole-mount (3D) methods have been recently developed to investigate DRG morphology and neurochemistry without tissue slicing, giving the opportunity to study the intimate relationship between SGCs and senso

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.

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.

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.

The onset of chemotherapy-induced peripheral neurotoxicity (CIPN) is a leading cause of the dose reduction or discontinuation of cancer treatment due to sensory symptoms. Paclitaxel (PTX) can cause painful peripheral neuropathy, with a negative impact on cancer survivors’ quality of life. While recent studies have shown that neuroinflammation is involved in PTX-induced peripheral neurotoxicity (PIPN), the pathophysiology of this disabling side effect remains largely unclear and no effective therapies are available. Therefore, here we investigated the effects of human intravenous immunoglobulin (IVIg) on a PIPN rat model. PTX-treated rats showed mechanical allodynia and neurophysiological alterations consistent with a severe sensory axonal polyneuropathy. In addition, morphological evaluation showed a reduction of intra-epidermal nerve fiber (IENF) density and evidenced axonopathy with macrophage infiltration, which was more prominent in the distal segment of caudal nerves. Three weeks after the last PTX injection, mechanical allodynia was still present in PTX-treated rats, while the full recovery in the group of animals co-treated with IVIg was observed. At the pathological level, this behavioral result was paralleled by prevention of the reduction in IENF density induced by PTX in IVIg co-treated rats. These results suggest that the immunomodulating effect of IVIg co-treatment can alleviate PIPN neurotoxic manifestations, probably through a partial reduction of neuroinflammation.

Glioblastoma is an extremely heterogeneous disease. Treatment failure and tumor recurrence primarily reflect the presence in the tumor core (TC) of the glioma stem cells (GSCs), and secondly the contribution, still to be defined, of the peritumoral brain zone (PBZ). Using the array-CGH platform, we deepened the genomic knowledge about the different components of GBM and we identified new specific biomarkers useful for new therapies. We firstly investigated the genomic profile of 20 TCs of GBM; then, for 14 cases and 7 cases, respectively, we compared these genomic profiles with those of the related GSC cultures and PBZ biopsies. The analysis on 20 TCs confirmed the intertumoral heterogeneity and a high percentage of copy number alterations (CNAs) in GBM canonical pathways. Comparing the genomic profiles of 14 TC-GSC pairs, we evidenced a robust similarity among the two samples of each patient. The shared imbalanced genes are related to the development and progression of cancer and in metabolic pathways, as shown by bioinformatic analysis using DAVID. Finally, the comparison between 7 TC-PBZ pairs leads to the identification of PBZ-unique alterations that require further investigation.

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 sta

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 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

Bortezomib (BTZ) is a proteasome inhibitor that represents the cornerstone in the treatment of multiple myeloma. Despite its efficacy, its clinical use is limited by severe painful peripheral neuropathy (BIPN) that occurs approximately in 50% of patients, impairing their life and representing a dose- imiting toxicity. There is increasing evidence that, instead of BTZ proteasome inhibition activity, BIPN may be due to BTZ off-targets. We focused our attention on tubulin and mitochondrial trafficking. In fact, on one hand, post-translational tubulin modification and Microtubule Associated Proteins MAPs) regulate microtubules (MTs) dynamics, fundamental for nervous system functions. On the other hand, axonal transport of mitochondria along the axons is essential for metabolic state and synaptic activity. Alterations of these two processes and modifications in their dynamics could lead to BIPN. In this work, we evaluated how these aspects take place in neurons isolated from adult mice dorsal root ganglia and treated with BTZ. MAP 2, delta2 tubulin a marker of hyperstable MTs) and tubulin acetylation were assessed through western blot analysis while the axonal mitochondrial trafficking as evaluated by time lapse confocal microscopy and kymograph analysis. We investigated the MTs stabilization by MAPs and by posttranslational modifications of tubulin and we found that BTZ treatment induced alterations of MAP2, delta2 tubulin and acetylated tubulin proteins quantity. After treatment for 24h with BTZ we also observed a loss of mitochondrial motility and dose-dependent reduction of transport speed, but no alterations were observed on the direction of the movement. Our results demonstrated that BTZ affect both mitochondrial trafficking and tubulin cytoskeleton in sensory neurons. Moreover, our data There is increasing evidence that, instead of BTZ proteasome inhibition activity, BIPN may be due to BTZ off-targets. We focused our attention on tubulin and mitochondrial traffick

Oxaliplatin (OHP) is an antineoplastic compound able to induce peripheral neurotoxicity. Oxidative stress has been suggested to be a key factor in the development of OHP‐related peripheral neurotoxicity. Mangafodipir, a contrast agent possessing mitochondrial superoxide dismutase (MnSOD)‐mimetic activity, has been tested as a cytoprotector in chemotherapy‐induced peripheral neurotoxicity (CIPN). Calmangafodipir (PledOx®) has even better therapeutic activity. We investigated a BALB/c mouse model of OHP‐related CIPN and the effects of the pre‐treatment of calmangafodipir (2.5, 5, or 10 mg/kg intravenously) on sensory perception, and we performed a pathological study on skin biopsies to assess intraepidermal nerve fiber (IENF) density. At the end of the treatments, OHP alone or in pre‐treatment with calmangafodipir 2.5 and 10 mg/kg, induced mechanical allodynia and cold thermal hyperalgesia, but calmangafodipir 5 mg/kg prevented these effects. Accordingly, OHP alone or in pre‐treatment with calmangafodipir 2.5 and 10 mg/kg, induced a significant reduction in IENF density, but calmangafodipir 5 mg/kg prevented this reduction. These results confirm a protective effect of calmangafodipir against OHP‐induced small fiber neuropathy. Interestingly, these results are in agreement with previous observations suggesting a U‐shaped effect of calmangafodipir, with the 10 mg/kg dose less effective than the lower doses.

Chemotherapy-induced peripheral neurotoxicity represents one of the most relevant dose-limiting side effects that can affect cancer patients treated with the common antineoplastic agents. Since the severity of neurotoxicity often leads to dose reduction or early cessation of chemotherapy, the investigation of molecular mechanisms underlying chemotherapy-induced peripheral neurotoxicity is an urgent clinical need in order to better understand its physiopathology and find effective strategies for neuroprotection. Several in vivo preclinical models of chemotherapy-induced peripheral neurotoxicity have been developed but a great variability in mouse strain, dose, route of administration of the drug, treatment schedule and assessment of neurotoxicity is observed between the different published studies making difficult the comparison and interpretation of their results. In many of these studies only behavioural tests are used as outcome measures, while possible neurophysiological and neuropathological changes are not evaluated. In this study, focused on experimental oxaliplatin-induced peripheral neurotoxicity, we reproduced and compared four mouse models with very different drug dose (low or high dose-intensity) and treatment schedules (short or long-term treatment), selected from the literature. Using a multimodal assessment based on behavioural, neurophysiological and neuropathological methods, we evidenced remarkable differences in the results obtained in the selected animal models. This work suggests the importance of a multimodal approach including extensive pathological investigation to confirm the behavioural results.