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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.

Recurrent somatic mutations in ETNK1 (Ethanolamine-Kinase-1) were identified in several myeloid malignancies and are responsible for a reduced enzymatic activity. Here, we demonstrate in primary leukemic cells and in cell lines that mutated ETNK1 causes a significant increase in mitochondrial activity, ROS production, and Histone H2AX phosphorylation, ultimately driving the increased accumulation of new mutations. We also show that phosphoethanolamine, the metabolic product of ETNK1, negatively controls mitochondrial activity through a direct competition with succinate at mitochondrial complex II. Hence, reduced intracellular phosphoethanolamine causes mitochondria hyperactivation, ROS production, and DNA damage. Treatment with phosphoethanolamine is able to counteract complex II hyperactivation and to restore a normal phenotype.

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.

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