Valentina Alda Carozzi

Bortezomib-induced painful neuropathy in rats: a behavioral, neurophysiological and pathological study in rats

Bortezomib is a proteasome inhibitor showing strong antitumor activity against many tumors, primarily multiple myeloma. Bortezomib‐induced neuropathic pain is the main side effect and the dose‐limiting factor of the drug in clinical practice. In order to obtain a pre‐clinical model to reproduce the characteristic pain symptoms in bortezomib‐treated patients, we developed an animal model of bortezomib‐induced nociceptive sensory neuropathy. In this study, bortezomib (0.15 or 0.20mg/kg) was administered to Wistar rats three times/week for 8 weeks, followed by a 4 week follow‐up period. At the end of the treatment period a significant decrease in weight gain was observed in the treated groups vs. controls, and hematological and histopathological parameters were evaluated. After the treatment period, both doses of bortezomib induced a severe reduction in nerve conduction velocity and demonstrated a dose‐cumulative effect of the drug. The sensory behavioral assessment showed the onset of mechanical allodynia, while no effect on thermal perception was observed.

Multimodal assessment of painful peripheral neuropathy induced by chronic oxaliplatin-based chemotherapy in mice

BackgroundA major clinical issue affecting 10-40% of cancer patients treated with oxaliplatin is severe peripheral neuropathy with symptoms including cold sensitivity and neuropathic pain. Rat models have been used to describe the pathological features of oxaliplatin-induced peripheral neuropathy; however, they are inadequate for parallel studies of oxaliplatins antineoplastic activity and neurotoxicity because most cancer models are developed in mice. Thus, we characterized the effects of chronic, bi-weekly administration of oxaliplatin in BALB/c mice. We first studied oxaliplatins effects on the peripheral nervous system by measuring caudal and digital nerve conduction velocities (NCV) followed by ultrastructural and morphometric analyses of dorsal root ganglia (DRG) and sciatic nerves. To further characterize the model, we examined nocifensive behavior and central nervous system excitability by in vivo electrophysiological recording of spinal dorsal horn (SDH) wide dynamic range neurons in oxaliplatin-treated miceResultsWe found significantly decreased NCV and action potential amplitude after oxaliplatin treatment along with neuronal atrophy and multinucleolated DRG neurons that have eccentric nucleoli. Oxaliplatin also induced significant mechanical allodynia and cold hyperalgesia, starting from the first week of treatment, and a significant increase in the activity of wide dynamic range neurons in the SDH.ConclusionsOur findings demonstrate that chronic treatment with oxaliplatin produces neurotoxic changes in BALB/c mice, confirming that this model is a suitable tool to conduct further mechanistic studies of oxaliplatin-related antineoplastic activity, peripheral neurotoxicity and pain. Further, this model can be used for the preclinical discovery of new neuroprotective and analgesic compounds.

Comparison of Neuropathy-Inducing Effects of Eribulin Mesylate, Paclitaxel, and Ixabepilone in Mice

Chemotherapy-induced neurotoxicity is a significant problem associated with successful treatment of many cancers. Tubulin is a well-established target of antineoplastic therapy; however, tubulin-targeting agents, such as paclitaxel and the newer epothilones, induce significant neurotoxicity. Eribulin mesylate, a novel microtubule-targeting analogue of the marine natural product halichondrin B, has recently shown antineoplastic activity, with relatively low incidence and severity of neuropathy, in metastatic breast cancer patients. The mechanism of chemotherapy-induced neuropathy is not well understood. One of the main underlying reasons is incomplete characterization of pathology of peripheral nerves from treated subjects, either from patients or preclinically from animals. The current study was conducted to directly compare, in mice, the neuropathy-inducing propensity of three drugs: paclitaxel, ixabepilone, and eribulin mesylate. Because these drugs have different potencies and pharmacokinetics, we compared them on the basis of a maximum tolerated dose (MTD). Effects of each drug on caudal and digital nerve conduction velocity, nerve amplitude, and sciatic nerve and dorsal root ganglion morphology at 0.25 × MTD, 0.5 × MTD, 0.75 × MTD, and MTD were compared. Paclitaxel and ixabepilone, at their respective MTDs, produced significant deficits in caudal nerve conduction velocity, caudal amplitude and digital nerve amplitudes, as well as moderate to severe degenerative pathologic changes in dorsal root ganglia and sciatic nerve. In contrast, eribulin mesylate produced no significant deleterious effects on any nerve conduction parameter measured and caused milder, less frequent effects on morphology. Overall, our findings indicate that eribulin mesylate induces less neuropathy in mice than paclitaxel or ixabepilone at equivalent MTD-based doses.

Neurophysiological and neuropathological characterization of new murine models of chemotherapy-induced chronic peripheral neuropathies

Cisplatin, paclitaxel and bortezomib belong to some of the most effective families of chemotherapy drugs for solid and haematological cancers. Epothilones represent a new family of very promising antitubulin agents. The clinical use of all these drugs is limited by their severe peripheral neurotoxicity. Several in vivo rat models have reproduced the characteristics of the peripheral neurotoxicity of these drugs. However, since only a very limited number of cancer types can be studied in immunocompetent rats, these animal models do not represent an effective way to evaluate, at the same time, the antineoplastic activity and the neurotoxic effects of the anticancer compounds. In this study, we characterized the neurophysiological impairment induced by chronic chemotherapy treatment in BALB/c mice, a strain suitable for assessing the activity of anticancer treatments. At the end of a 4-week period of treatment with cisplatin, paclitaxel, epothilone-B or bortezomib, sensory and sensory/motor nerve conduction velocities (NCV) were determined in the caudal and digital nerves and dorsal root ganglia (DRG) and sciatic nerves were collected for histopathological analysis. The electrophysiological studies revealed that all the compounds caused a statistically significant reduction in the caudal NCV, while impairment of the digital NCV was less severe. This functional damage was confirmed by the histopathological observations evidencing axonal degeneration in the sciatic nerve induced by all the drugs associated with pathological changes in DRG induced only by cisplatin and bortezomib. These results confirm the possibility to use our models to combine the study of the antineoplastic activity of anticancer drugs and of their toxic effects on the peripheral nervous system in the BALB/c mouse strain.

Neuroprotective effects of a ligand of translocator protein-18kDa (Ro5-4864) in experimental diabetic neuropathy

Peripheral neuropathy represents an important complication of diabetes involving a spectrum of structural, functional and biochemical alterations in peripheral nerves. Recent observations obtained in our laboratory have shown that the levels of neuroactive steroids present in the sciatic nerve of rat raised diabetic by a single injection of streptozotocin (STZ) are reduced and that, in the same experimental model, treatment with neuroactive steroids, such as progesterone, testosterone and their derivatives show neuroprotective effects. On this basis, an interesting therapeutic strategy could be to increase the levels of neuroactive steroids directly in the nervous system. With this perspective, ligands of translocator protein-18 kDa (TSPO) may represent an interesting option. TSPO is mainly present in the mitochondrial outer membrane, where it promotes the translocation of cholesterol to the inner mitochondrial membrane, and, as demonstrated in other cellular systems, it allows the transformation of cholesterol into pregnenolone and the increase of steroid levels. In the diabetic model of STZ rat, we have here assessed whether treatment with Ro5-4864 (i.e., a ligand of TSPO) could increase the low levels of neuroactive steroids in sciatic nerve and consequently to be protective in this experimental model. Data obtained by liquid chromatography-tandem mass spectrometry show that treatment with Ro5-4864 was able to significantly stimulate the low levels of pregnenolone, progesterone and dihydrotestosterone observed in the sciatic nerves of diabetic rats. The treatment with Ro5-4864 also counteracted the impairment of NCV and thermal threshold, restored skin innervation density and P0 mRNA levels, and improved Na+,K+-ATPase activity. In conclusion, data here reported show for the first time that a TSPO ligand, such as Ro5-4864, is effective in reducing the severity of diabetic neuropathy through a local increase of neuroactive steroid levels.

Docetaxel-induced peripheral neuropathy: protective effects of dihydroprogesterone and progesterone in an experimental model

Abstract  Peripheral neurotoxicity is a frequent complication limiting docetaxel chemotherapy in patients with cancer. We developed an experimental model that closely mimics the course of neuropathy in patients, aiming to investigate both the mechanisms of neurotoxicity at biochemical, functional and morphological levels and the potential neuroprotective role of neuroactive steroids. We demonstrated that treatment with dihydroprogesterone (DHP) or progesterone (P) counteracts docetaxel‐induced neuropathy, preventing nerve conduction and thermal threshold changes, and degeneration of skin nerves in the foodpad. Neuroactive steroids also counteract the changes in gene expression of several myelin proteins and calcitonin gene‐related peptide induced by docetaxel in sciatic nerve and lumbar spinal cord, respectively. Most nerve abnormalities observed during the treatment with docetaxel spontaneously recovered after drug withdrawal, similarly to what occurs in patients. However, results of midterm follow‐up experiments indicated that animals cotreated with DHP or P have a faster recovery of the neuropathy compared with docetaxel‐treated rats. Our study confirmed that neuroactive steroids exert a protective effect on peripheral nerves at different levels, suggesting that they might represent a new therapeutic frontier for patients with chemotherapy‐induced neuropathy.

Bortezomib-induced painful peripheral neuropathy: an electrophysiological, behavioral, morphological and mechanistic study in the mouse

Bortezomib is the first proteasome inhibitor with significant antineoplastic activity for the treatment of relapsed/refractory multiple myeloma as well as other hematological and solid neoplasms. Peripheral neurological complications manifesting with paresthesias, burning sensations, dysesthesias, numbness, sensory loss, reduced proprioception and vibratory sensitivity are among the major limiting side effects associated with bortezomib therapy. Although bortezomib-induced painful peripheral neuropathy is clinically easy to diagnose and reliable models are available, its pathophysiology remains partly unclear. In this study we used well-characterized immune-competent and immune-compromised mouse models of bortezomib-induced painful peripheral neuropathy. To characterize the drug-induced pathological changes in the peripheral nervous system, we examined the involvement of spinal cord neuronal function in the development of neuropathic pain and investigated the relevance of the immune response in painful peripheral neuropathy induced by bortezomib. We found that bortezomib treatment induced morphological changes in the spinal cord, dorsal roots, dorsal root ganglia (DRG) and peripheral nerves. Neurophysiological abnormalities and specific functional alterations in Aδ and C fibers were also observed in peripheral nerve fibers. Mice developed mechanical allodynia and functional abnormalities of wide dynamic range neurons in the dorsal horn of spinal cord. Bortezomib induced increased expression of the neuronal stress marker activating transcription factor-3 in most DRG. Moreover, the immunodeficient animals treated with bortezomib developed a painful peripheral neuropathy with the same features observed in the immunocompetent mice. In conclusion, this study extends the knowledge of the sites of damage induced in the nervous system by bortezomib administration. Moreover, a selective functional vulnerability of peripheral nerve fiber subpopulations was found as well as a change in the electrical activity of wide dynamic range neurons of dorsal horn of spinal cord. Finally, the immune response is not a key factor in the development of morphological and functional damage induced by bortezomib in the peripheral nervous system.

Glutamate carboxypeptidase inhibition reduces the severity of chemotherapy-induced peripheral neurotoxicity in rat.

Chemotherapy is the most common method to treat cancer. The use of certain antineoplastic drugs, however, is associated with the development of peripheral neuropathy that can be dose-limiting. Excitotoxic glutamate release, leading to excessive glutamatergic neurotransmission and activation of N-methyl-d-aspartate (NMDA) receptors, is associated with neuronal damage and death in several nervous system disorders. N-Acetyl-aspartyl-glutamate (NAAG) is an abundant neuropeptide widely distributed in the central and peripheral nervous system which is physiologically hydrolyzed by the enzyme glutamate carboxypeptidase into N-Acetyl-aspartyl (NAA) and glutamate. Pharmacological inhibition of glutamate carboxypeptidase results in decreased glutamate and increased endogenous NAAG and has been shown to provide neuroprotection in several preclinical models. Here, we report the neuroprotective effect of an orally available glutamate carboxypeptidase inhibitor on three well-established animal models of chemotherapy (cisplatin, paclitaxel, bortezomib)-induced peripheral neuropathy. In all cases, glutamate carboxypeptidase inhibition significantly improved the chemotherapy-induced nerve conduction velocity deficits. In addition, morphological and morphometrical alterations induced by cisplatin and bortezomib in dorsal root ganglia (DRG) were improved by glutamate carboxypeptidase inhibition. Our data support a novel approach for the treatment of chemotherapy-induced peripheral neuropathy.

Expression and distribution of 'high affinity' glutamate transporters GLT1, GLAST, EAAC1 and of GCPII in the rat peripheral nervous system.

l‐Glutamate is one of the major excitatory neurotransmitters in the mammalian central nervous system, but recently it has been shown to have a role also in the transduction of sensory input at the periphery, and in particular in the nociceptive pathway. An excess of glutamate is implicated in cases of peripheral neuropathies as well. Conventional therapeutic approaches for treating these diseases have focused on blocking glutamate receptors with small molecules or on reducing its synthesis of the receptors through the inhibition of glutamate carboxypeptidase II (GCPII), the enzyme that generates glutamate. In vivo studies have demonstrated that the pharmacological inhibition of GCPII can either prevent or treat the peripheral nerve changes in both BB/Wor and chemically induced diabetes in rats. In this study, we characterized the expression and distribution of glutamate transporters GLT1, GLAST, EAAC1 and of the enzyme GCPII in the peripheral nervous system of female Wistar rats. Immunoblotting results demonstrated that all glutamate transporters and GCPII are present in dorsal root ganglia (DRG) and the sciatic nerve. Immunofluorescence localization studies revealed that both DRG and sciatic nerves were immunopositive for all glutamate transporters and for GCPII. In DRG, satellite cells were positive for GLT1 and GCPII, whereas sensory neurons were positive for EAAC1. GLAST was localized in both neurons and satellite cells. In the sciatic nerve, GLT1 and GCPII were expressed in the cytoplasm of Schwann cells, whereas GLAST and EAAC1 stained the myelin layer. Our results give for the first time a complete characterization of the glutamate transporter system in the peripheral nervous system. Therefore, they are important both for understanding glutamatergic signalling in the PNS and for establishing new strategies to treat peripheral neuropathies.

CR4056, a new analgesic I2 ligand, is highly effective against bortezomib-induced painful neuropathy in rats

Although bortezomib (BTZ) is the frontline treatment for multiple myeloma, its clinical use is limited by the occurrence of painful peripheral neuropathy, whose treatment is still an unmet clinical need. Previous studies have shown chronic BTZ administration (0.20 mg/kg intravenously three times a week for 8 weeks) to female Wistar rats induced a peripheral neuropathy similar to that observed in humans. In this animal model of BTZ-induced neurotoxicity, the present authors evaluated the efficacy of CR4056, a novel I2 ligand endowed with a remarkable efficacy in several animal pain models. CR4056 was administered in a wide range of doses (0.6–60 mg/kg by gavage every day for 2–3 weeks) in comparison with buprenorphine (Bupre) (28.8 μg/kg subcutaneously every day for 2 weeks) and gabapentin (Gaba) (100 mg/kg by gavage every day for 3 weeks). Chronic administration of BTZ reduced nerve conduction velocity and induced allodynia. CR4056, Bupre, or Gaba did not affect the impaired nerve conduction velocity. Conversely, CR4056 dose-dependently reversed BTZ-induced allodynia (minimum effective dose 0.6 mg/kg). The optimal dose found, 6 mg/kg, provided a constant pain relief throughout the treatment period and without rebound after suspension, being effective when coadministered with BTZ, starting before or after allodynia was established, or when administered alone after BTZ cessation. A certain degree of tolerance was seen after 7 days of administration, but only at the highest doses (20 and 60 mg/kg). Bupre was effective only acutely, since tolerance was evident from the fourth day onwards. Gaba showed a significant activity only at the fourth day of treatment. CR4056, over the range of concentrations of 3–30 μM, was unable to hinder BTZ cytotoxicity on several tumor cell lines, which could indicate that this substance does not directly interfere with BTZ antitumor activity. Therefore, CR4056 could represent a new treatment option for BTZ-induced neuropathic pain.