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Cristina Meregalli,1 Cecilia Ceresa,1 Annalisa Canta,1 Valentina Alda Carozzi,1 Alessia Chiorazzi,1 Barbara Sala,1 Norberto Oggioni,1 Marco Lanza,2 Ornella Letar,i2 Flora Ferrari,2 Federica Avezza,1 Paola Marmiroli,1 GianFranco Caselli,2 Guido Cavaletti11Department of Neuroscience and Biomedical Technologies, University of Milan-Bicocca, 2Pharmacology and Toxicology Department, Rottapharm | Madaus Research Center, Monza, ItalyAbstract: 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.Keywords: imidazoline I2 receptor ligand, antinociception, allodynia, neuropathic pain, bortezomib

Animal models of human diseases affecting the peripheral nervous system are widely used to assess the pathogenesis of neurotoxicity and to compare the effect of new agents. Several behavioural, pathological and neurophysiological methods have been used, and each has advantages and disadvantages. A major goal in the study of neurotoxicity would be to assess the damage in the same way in animal models and in humans. In this study we correlated the neurophysiological results obtained in normal rats and in rats treated with cisplatin 2 mg/kg q3d × 8 with the density of intraepidermal fibers (IEF) obtained in skin biopsy specimens. The aim was to investigate the possible role of a minimally invasive procedure such as skin biopsy as an alternative method to assess the peripheral neurotoxicity of antineoplastic drugs. The nerve conduction velocity (NCV) in the tail nerve was assessed in thirty-six young adult female Wistar rats which were left untreated, or treated with erythropoietin (EPO), cisplatin (CDDP) or EPO + CDDP. CDDP and CDDP + EPO-treated rats had a significantly reduced NCV vs. age-matched untreated rats. At sacrifice, skin specimens were obtained. The density of IEF was calculated by 2 independent blinded examiners and the correlation existing between NCV and IEF was highly significant (r = 0.670, p < 0.001). This preliminary result suggests that IEF should be evaluated in other animal models and might represent a useful tool to study peripheral neurotoxicity also in humans. [ABSTRACT FROM AUTHOR]

The pathogenesis of the neurotoxicity of most antineoplastic drugs is unknown. Recent reports suggest that changes in the circulating levels of nerve growth factor (NGF) might be related to the dorsal root ganglia sensory neuron damage induced by cisplatin (CDDP), the first member of a family of widely used and very effective platinum-derived anticancer agents. Using a well-characterized model of CDDP neurotoxicity, we demonstrated that the NGF circulating level decreased during chronic CDDP administration in close accordance with the clinical course and returned to normal levels after recovery from the neurotoxic damage. Moreover, these changes were restricted to NGF and did not involve other trophic factors of the same neurotrophin family. Our findings are in agreement with previous in vitro and in vivo results and further suggest that NGF plays a specific role in the course of CDDP-induced primary sensory neuron damage. [Copyright &y& Elsevier]

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.

Chemotherapy-induced peripheral neurotoxicity is a common dose-limiting side effect of several cancer chemotherapeutic agents, and no effective therapies exist. Here we constructed a systems pharmacology model of intracellular signaling in peripheral neurons to identify novel drug targets for preventing peripheral neuropathy associated with proteasome inhibitors. Model predictions suggested the combinatorial inhibition of TNFα, NMDA receptors, and reactive oxygen species should prevent proteasome inhibitor-induced neuronal apoptosis. Dexanabinol, an inhibitor of all three targets, partially restored bortezomib-induced reduction of proximal action potential amplitude and distal nerve conduction velocity in vitro and prevented bortezomib-induced mechanical allodynia and thermal hyperalgesia in rats, including a partial recovery of intraepidermal nerve fiber density. Dexanabinol failed to restore bortezomib-induced decreases in electrophysiological endpoints in rats, and it did not compromise bortezomib anti-cancer effects in U266 multiple myeloma cells and a murine xenograft model. Owing to its favorable safety profile in humans and preclinical efficacy, dexanabinol might represent a treatment option for bortezomib-induced neuropathic pain., Competing Interests: MM is co-founder and CSO of AxoSim, Inc. DM is president and CEO of Enhanced Pharmacodynamics, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bloomingdale, Meregalli, Pollard, Canta, Chiorazzi, Fumagalli, Monza, Pozzi, Alberti, Ballarini, Oggioni, Carlson, Liu, Ghandili, Ignatowski, Lee, Moore, Cavaletti and Mager.)

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.

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., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The comments sent by Stehr, Lundstom and Karlsson with reference to our article "Calmangafodipir reduces sensory alterations and prevents intraepidermal nerve fiber loss in a mouse model of oxaliplatin-induced peripheral neurotoxicity" are very interesting, since they suggest possible mechanisms of action of the compound, which might contribute to its protective action [...].

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.

Oxaliplatin (OHP) Induced Peripheral Neurotoxicity (OIPN) is one of the dose-limiting toxicities of the drug and these adverse effects limit cancer therapy with L-OHP, used for colorectal cancer treatment. Acute neurotoxicity consists of symptoms that are the hallmarks of a transient axonal hyperexcitability; chronic neurotoxicity has a clinical picture compatible with a length-dependent sensory neuropathy. Acute OIPN pathogenesis has been linked to sodium voltage-operated channels (Na + VOC) dysfunction and it has been advocated as a possible predisposing factor to chronic neurotoxicity. We tested if topiramate (TPM), a well-known Na + VOC modulator, was able to modify acute as well as chronic OIPN. The project was divided into two parts. In Experiment 1 we tested by means of Nerve Excitability Testing (NET) a cohort of female Wistar rats to assess TPM effects after a single OHP administration (5 mg/kg, iv). In Experiment 2 we assessed TPM effects after chronic OHP treatment (5 mg/kg, 2qw4ws, iv) using NET, nerve conduction studies (NCS), behavioral tests and neuropathology (caudal nerve morphometry and morphology and Intraepidermal Nerve Fiber [IENF] density). In Experiment 1 TPM was able to prevent OHP effects on Na + VOC: OHP treatment induced a highly significant reduction of the sensory nerve's threshold, during the superexcitability period (p-value = 0.008), whereas TPM co-administration prevented this effect. In Experiment 2 we verified that TPM was able to prevent not only acute phenomena, but also to completely prevent chronic OIPN. This latter observation was supported by a multimodal approach: in fact, only OHP group showed altered findings compared to CTRL group at a neurophysiological (proximal caudal nerve sensory nerve action potential [SNAP] amplitude, p-value = 0.001; distal caudal nerve SNAP amplitude, p-value<0.001, distal caudal nerve sensory conduction velocity, p-value = 0.04), behavioral (mechanical threshold, p-value 0.003) and neuropathological levels (caudal nerve fibers density, p-value 0.001; IENF density, p-value <0.001). Our data show that TPM is a promising drug to prevent both acute and chronic OIPN. These findings have a high translational potential, since they were obtained using outcome measures that match clinical practice and TPM is already approved for clinical use being free from detrimental interaction with OHP anticancer properties., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Multiple cell signaling pathways are implicated in the development, progression, and persistence of cisplatin-induced peripheral neuropathy. Although advances have been made in terms of understanding specific neurotoxic mechanisms, there are few predictive factors identified that can help inform the clinician approach to symptom prevention or management., Objective: We investigate the differential sensitivity to cisplatin-induced peripheral neuropathy and examine the contribution of dorsal root ganglion (DRG) transcriptional profiles across two inbred strains of mice., Methods: Cisplatin (4 mg/kg intraperitoneal or vehicle control) was administered twice a week for 4 weeks to adult female C57BL/6J and A/J mice-the C57BL/6J strain of mice characterized by a robust mechanical allodynia and the A/J with a mild largely resistant allodynia phenotype. Peripheral nerve conduction velocities (NCVs), electrophysiological evaluation of wide dynamic range (WDR) neurons, morphological examination of DRG neurons, and microarray analysis of spinal cord tissues were compared across the 4 weeks., Results: The A/J strain presents with an early, mild nocifensive response to cisplatin with reduced neuronal activity in WDR neurons and small changes in cross-sectional nucleus size in DRG neurons at 4 weeks. The more nocifensive-sensitive C57BL/6J strain presents with no early changes in WDR neuron responsiveness; however, there were significant changes in DRG size. Both strains demonstrate a drop in NCV after 4 weeks of treatment, with the greatest reduction present in the A/J strain. Transcriptome data implicate neuroimmune modulation in the differential response to cisplatin in the DRGs of A/J and C57BL/6J mice., Discussion: Nocifensive responses in both strains implicate involvement of small myelinated and unmyelinated fibers in neurotoxic cisplatin response, whereas reductions in NCV reflect involvement of the largest myelinated fibers in the peripheral nerves. Microarray data analysis identifies neuropathy-relevant gene sets with differential activation of pathways, suggesting a role for antigen presentation in the differential neurotoxic response to cisplatin across strains. Further research is indicated to determine the relative contributions of each of these potential pathological mechanisms to both the neurotoxic response to cisplatin and to the potential for targeted therapy.

Background: Chemotherapy-induced peripheral neurotoxicity (CIPN) is a severe adverse effect in patients receiving antitumor agents, and no effective treatment is available. Although the mechanisms responsible for the development of CIPN are poorly understood, recent findings make neuroinflammation an attractive target to be investigated, particularly when neuropathic pain is a prominent feature such as after bortezomib administration. The aim of our study was to evaluate the effect of intravenous immunoglobulins (IVIg) delivery in chronic CIPN. The related neuro-immune aspects were investigated in a well-characterized rat model of bortezomib-induced peripheral neurotoxicity (BIPN)., Methods: After determination of a suitable schedule based on a preliminary pharmacokinetic pilot study, female Wistar rats were treated with IVIg 1 g/kg every 2 weeks. IVIg treatment was started at the beginning of bortezomib administration ("preventive" schedule), or once BIPN was already ensued after 4 weeks of treatment ("therapeutic" schedule). Neurophysiological and behavioral studies were performed to assess the extent of painful peripheral neurotoxicity induced by bortezomib, and these functional assessments were completed by pathologic examination of peripheral nerves and intraepidermal nerve fiber quantification (IENF). The role of the innate immune response in BIPN was investigated by immunochemistry characterization of macrophage infiltration in peripheral nerves., Results: Both schedules of IVIg administration were able to significantly reduce bortezomib-induced heat and mechanical allodynia. Although these changes were not evidenced at the neurophysiological examination of peripheral nerves, they behavioral effects were paralleled in the animals treated with the preventive schedule by reduced axonopathy in peripheral nerves and significant protection from loss of IENF. Moreover, IVIg administration was very effective in reducing infiltration in peripheral nerves of macrophages with the M1, pro-inflammatory phenotype., Conclusion: Our results suggest a prominent role of neuroinflammation in BIPN and that IVIg might be considered as a possible safe and effective therapeutic option preventing M1 macrophage infiltration. However, since neuropathic pain is frequent also in other CIPN types, it also indicates the need for further investigation in other forms of CIPN.

Type-1 Diabetes is generally treated with exogenous insulin administration. Despite treatment, a very common long term consequence of diabetes is the development of a disabling and painful peripheral neuropathy. The transplantation of pancreatic islets is an advanced alternative therapeutic approach, but its clinical application is still very limited, mainly because of the great number of islets required to complete the procedure and of their short-term survival. An intriguing method to improve the performance of pancreatic islets transplantation is the co-transplantation of Mesenchymal Stem Cells (MSCs), adult stem cells already known to support the survival of different cellular populations. In this proof-of-concept study, we demonstrated using an in vivo model of diabetes, the ability of allogenic MSCs to reduce the number of pancreatic islets necessary to achieve glycemic control in diabetic rats, and overall their positive effect on diabetic neuropathy, with the reduction of all the neuropathic signs showed after disease induction. The cutback of the pancreatic islet number required to control glycemia and the regression of the painful neuropathy make MSC co-transplantation a very promising tool to improve the clinical feasibility of pancreatic islet transplantation for diabetes treatment., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This study is aimed at describing the changes occurring in the entire peripheral nervous system sensory pathway along a 2-year observation period in a cohort of C57BL/6 mice. The neurophysiological studies evidenced significant differences in the selected time points corresponding to childhood, young adulthood, adulthood, and aging (i.e., 1, 7, 15, and 25 months of age), with a parabolic course as function of time. The pathological assessment allowed to demonstrate signs of age-related changes since the age of 7 months, with a remarkable increase in both peripheral nerves and dorsal root ganglia at the subsequent time points. These changes were mainly in the myelin sheaths, as also confirmed by the Rotating-Polarization Coherent-Anti-stokes-Raman-scattering microscopy analysis. Evident changes were also present at the morphometric analysis performed on the peripheral nerves, dorsal root ganglia neurons, and skin biopsies. This extensive, multimodal characterization of the peripheral nervous system changes in aging provides the background for future mechanistic studies allowing the selection of the most appropriate time points and readouts according to the investigation aims., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aim: Nowadays, there is a great interest in the therapeutic potential of sigma1 receptor ligands for treating different CNS pathologies. Our previous investigations led to identify (R)-RC-33 as a potent and selective S1R agonist., Results: Herein, we report the gram-scale synthesis, pharmacokinetic profile and CNS distribution of (R)-RC-33 in the mouse to determine the most suitable dosage schedule for in vivo administration. For comparative purposes, the same experiments were also performed with PRE-084, the most widely used S1R agonist commonly in pharmacological experiments., Discussion: (R)-RC-33 shows a similar pharmacokinetic profile and a better CNS distribution when compared with PRE-084., Conclusion: (R)-RC-33 may be a promising candidate for in vivo studies in animal models of neurodegenerative diseases.

1-Deoxysphingolipids (1-deoxySLs) are atypical neurotoxic sphingolipids that are formed by the serine-palmitoyltransferase (SPT). Pathologically elevated 1-deoxySL concentrations cause hereditary sensory and autonomic neuropathy type 1 (HSAN1), an axonal neuropathy associated with several missense mutations in SPT. Oral L-serine supplementation suppressed the formation of 1-deoxySLs in patients with HSAN1 and preserved nerve function in an HSAN1 mouse model. Because 1-deoxySLs also are elevated in patients with type 2 diabetes mellitus, L-serine supplementation could also be a therapeutic option for diabetic neuropathy (DN). This was tested in diabetic STZ rats in a preventive and therapeutic treatment scheme. Diabetic rats showed significantly increased plasma 1-deoxySL concentrations, and L-serine supplementation lowered 1-deoxySL concentrations in both treatment schemes (P < 0.0001). L-serine had no significant effect on hyperglycemia, body weight, or food intake. Mechanical sensitivity was significantly improved in the preventive (P < 0.01) and therapeutic schemes (P < 0.001). Nerve conduction velocity (NCV) significantly improved in only the preventive group (P < 0.05). Overall NCV showed a highly significant (P = 5.2E-12) inverse correlation with plasma 1-deoxySL concentrations. In summary, our data support the hypothesis that 1-deoxySLs are involved in the pathology of DN and that an oral L-serine supplementation could be a novel therapeutic option for treating DN., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Cisplatin, paclitaxel and bortezomib are effective chemotherapy drugs in cancer treatment. However, they share severe peripheral neurotoxicity (PN) as one of their major dose-limiting side effects, often impairing cancer patients' quality of life and sometimes being permanent. Even if preclinical oncology is largely based on the use of immune-deficient mice, rodent models used to study the chemotherapy-induced PN are available only in immune-competent animals. In this study we characterized for the first time the PN induced by these chemotherapies through neurophysiological, behavioral, morphological and morphometric studies in athymic nude mice, a commonly employed strain in the preclinical oncology. The animals, divided into four groups, were chronically treated with cisplatin, paclitaxel or bortezomib once or twice a week for 4 or 6 weeks or were left untreated. These schedules were tolerated, neurotoxic and in the range of antineoplastic effectiveness. Despite similarities, differences in the features of PN were evident if compared with immune-competent models under comparable regimens of treatment. The results of this study may provide a basis for future combined analysis of antineoplastic and neurotoxic effects of chemotherapy in the same animals., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

The pathogenetic role of vascular endothelial growth factor (VEGF) in long-term retinal and kidney complications of diabetes has been demonstrated. Conversely, little is known in diabetic neuropathy. We examined the modulation of VEGF pathway at mRNA and protein level on dorsal root ganglion (DRG) neurons and Schwann cells (SC) induced by hyperglycaemia. Moreover, we studied the effects of VEGF neutralization on hyperglycemic DRG neurons and streptozotocin-induced diabetic neuropathy. Our findings demonstrated that DRG neurons were not affected by the direct exposition to hyperglycaemia, whereas showed an impairment of neurite outgrowth ability when exposed to the medium of SC cultured in hyperglycaemia. This was mediated by an altered regulation of VEGF and FLT-1 receptors. Hyperglycaemia increased VEGF and FLT-1 mRNA without changing their intracellular protein levels in DRG neurons, decreased intracellular and secreted protein levels without changing mRNA level in SC, while reduced the expression of the soluble receptor sFLT-1 both in DRG neurons and SC. Bevacizumab, a molecule that inhibits VEGF activity preventing the interaction with its receptors, restored neurite outgrowth and normalized FLT-1 mRNA and protein levels in co-cultures. In diabetic rats, it both prevented and restored nerve conduction velocity and nociceptive thresholds. We demonstrated that hyperglycaemia early affected neurite outgrowth through the impairment of SC-derived VEGF/FLT-1 signaling and that the neutralization of SC-secreted VEGF was protective both in vitro and in vivo models of diabetic neuropathy.

Bortezomib (BTZ) is the first proteasome inhibitor entered in clinical practice. Peripheral neuropathy is likely to be a class side effect of these drugs, although its severity is largely variable, and it deserves to be further investigated, since the mechanisms of BTZ-induced peripheral neurotoxicity (BiPN) are still unknown.   In our study, we investigated in vivo and in vitro possible pathogenic events relevant to BiPN using a well-established rat model, with particular reference to the extent of proteasome inhibition and the effects on α-tubulin polymerization in sciatic nerves and dorsal root ganglia specimens obtained from animals treated with chronic regimens at a dose of 0.2 mg/kg intravenously. The same assessments were also performed after a single injection. Moreover, these studies were replicated in vitro using embryonic DRG neurons exposed to 100 nM BTZ and adult DRG neurons exposed to 10-50 nM BTZ for 24 h and 48 h. A significant increase in the polymerized fraction of α-tubulin and prolonged proteasome inhibition were observed after the chronic BTZ treatment in vivo. Recovery to physiological levels was observed after a 4-week follow-up post-treatment period. Proteasome inhibition and increased α-tubulin polymerization were also observed following BTZ treatment of both embryonic and adult DRG neurons in vitro. Our in vivo results suggest that proteasome inhibition and alteration of tubulin dynamics contribute to BiPN. The in vitro systems here described reliably replicate the in vivo results, and might therefore be used for further mechanistic studies on the effects of proteasome inhibitors on neurons.

Background: Bortezomib is an anti-neoplastic drug acting against multiple myeloma but its use is associated with the onset of painful peripheral neuropathy. Tumor necrosis factor-α (TNFα) is associated with the development of neuropathic pain; several models have shown that the inactivation of TNFα leads to a reduction in pain stimuli perception. The aim of the present study was to analyze if the administration of an antibody against TNFα is able to prevent the development of bortezomib-induced neuropathic pain., Materials and Methods: Nerve conduction velocity was measured and a histopathological examination was performed to assess the extent of peripheral neuropathy. To study the onset of painful neuropathy, the response to mechanical or thermal stimuli was evaluated., Results: This study demonstrated that co-administration of an antibody against TNFα is able to prevent allodynia induced by bortezomib but does not reduce neuropathy., Conclusion: Targeting TNFα might be useful in limiting patients' discomfort during bortezomib therapy.

Islet transplantation is a poorly investigated long-term strategy for insulin replacement and for treatment of complications in patients with diabetes. We investigated whether islet transplantation and insulin treatment can relieve diabetic neuropathy and rescue the residual endogenous pancreatic β cells. We used a multimodal approach, with five groups of Sprague-Dawley rats studied for 8 months: control rats, diabetic rats, insulin-treated diabetic rats with moderate or mild hyperglycemia, and diabetic rats transplanted with microencapsulated islets. Islet transplantation normalized glycemia and increased body and muscle weight; it was also effective in reducing proteinuria and altered liver function. Transplantation significantly improved tail nerve conduction velocity, Na(+)-K(+)-ATPase activity, and morphological alterations in the sciatic nerve as evidenced by decrease in g-ratio; it also restored thermal and ameliorated mechanical nociceptive thresholds. Morphometric analysis of pancreas indicated a significant β-cell volume increase in transplanted rats, compared with mildly and moderately hyperglycemic rats. Thus, allogeneic islet transplantation had a positive systemic effect in diabetic rats and induced regression of the established neuropathy and restitution of the typical characteristics of the islets. These findings strongly reinforce the need for improving glycemic control, not only to reverse established diabetic complications but also to improve β-cell status in diabetic pancreas., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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.

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.

1-[(2-adamantyl)amino]acetyl-2-cyano-(S)-pyrrolidine, monohydrochloride (PKF275-055), a vildagliptin analog, is a novel, selective, potent, orally bioavailable, and long-acting dipeptidyl peptidase IV inhibitor. We studied the effect of PKF275-055 administration on the prevention, protection, and treatment of diabetic neuropathy in the streptozotocin-induced diabetic rat. PKF275-055 improved body and muscle weight. Oral glucose tolerance tests showed a marked improvement in glucose metabolism under all treatment schedules. When tested in prevention and protection experiments, PKF275-055 completely averted the decrease of Na⁺/K⁺-ATPase activity and partially counteracted the nerve conduction velocity (NCV) deficit observed in untreated diabetic rats but had no effects on abnormal mechanical and thermal sensitivity. When used in a therapeutic setting, PKF275-055 induced a significant correction in the alteration in Na⁺,K⁺-ATPase activity and NCV present in untreated diabetics. Diabetic rats developed mechanical hyperalgesia within 2 weeks after streptozotocin injection and exhibited significantly longer thermal response latencies. It is noteworthy that PKF275-055 treatment restored mechanical sensitivity thresholds by approximately 50% (p < 0.01) and progressively improved the alteration in thermal responsiveness. In conclusion, PKF275-055 showed an anabolic effect, improved oral glucose tolerance, and counteracted the alterations in Na⁺,K⁺-ATPase activity, NCV, and nociceptive thresholds in diabetic rats. The present data support a potential therapeutic effect of PKF275-055 in the treatment of rodent diabetic neuropathy.

Purpose: Alterations in mRNA for myelin proteins are reported in animal models of chemotherapy-induced peripheral neuropathies (CIPN); however, ultrastructural changes in aldehyde-fixed and plastic-embedded myelin are not evident by electron microscopy. Therefore, we used X-ray diffraction (XRD) to investigate more subtle changes in myelin sheath structure from unfixed nerves., Experimental Design: We used in vivo chronic animal models of CIPN in female Wistar rats, administering cisplatin (CDDP 2mg/kg, i.p. twice/week), paclitaxel (PT 10mg/kg, i.v. once/week) or bortezomib (0.20mg/kg, i.v. three times/week) over a total period of 4weeks. Animal weights were monitored, and tail nerve conduction velocity (NCV) was determined at the end of the treatments to assess the occurrence of peripheral neuropathy. Sciatic nerves were collected and the myelin structure was analyzed using electron microscopy (EM) and XRD., Results: All the rats treated with the chemotherapy agents developed peripheral neuropathy, as indicated by a decrease in NCV values; however, light and electron microscopy indicated no severe pathological alterations of the myelin morphology. XRD also did not demonstrate significant differences between sciatic nerves in treated vs. control rats with respect to myelin period, relative amount of myelin, membrane structure, and regularity of membrane packing., Conclusions: These results indicate that experimental peripheral neuropathy caused by CDDP, PT, and bortezomib-which are among the most widely used chemotherapy agents-does not significantly affect the structure of internodal myelin in peripheral nerve., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Taxanes, including docetaxel (DOCE), are severely neurotoxic, causing disabling peripheral neuropathy. Co-treatment with neuroprotective agents has been proposed to prevent or reverse this. Besides its hemopoietic effects, erythropoietin (EPO) has neuroprotective and neurotrophic properties and when administered systemically it has a wide range of neuroprotective action in animal models of nervous system damage, including cisplatin-induced peripheral neurotoxicity. The present study investigated the effects of EPO on chemotherapy-induced peripheral neurotoxicity (CINP) by DOCE in vivo and whether it interfered with tumor growth or antitumor activity. Female Fischer rats bearing 13762 mammary carcinoma were randomly divided into four groups: untreated, treated with EPO, DOCE, or DOCE + EPO. DOCE was given once a week (5 mg/kg, i.v.) and EPO three times a week (50 microg/kg i.p.), for 4 weeks. Three other groups of rats without tumors were left untreated or given DOCE or DOCE + EPO. The rats were observed for 4 weeks after treatment. CINP and neuroprotection were evaluated by measuring nociception, electrophysiological, and biochemical parameters. EPO protected against CINP, and tumor growth in EPO-treated rats was the same as in controls. EPO significantly improved the thermal threshold, tail nerve conduction velocity, and intra-epidermal nerve fiber density. These benefits lasted through the follow-up period and EPO speeded-up spontaneous recovery after treatment withdrawal. EPO did not impair DOCE antitumor activity. Since CINP induced by DOCE reproduces the clinical utility of taxane in humans, the findings reported might provide a basis for investigating EPO as a neuroprotective agent in patients receiving therapy with DOCE.

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. Sciatic nerves and dorsal root ganglia (DRG) were collected at the end of the 8-week treatment and at the end of the follow-up period. The pathological examination revealed a dose-dependent axonopathy of the unmyelinated fibers in nerves of treated animals. No pathological alteration in most of DRG satellite cells and neurons was observed. Therefore, this animal model may be useful for studying the neurotoxicity and pain onset mechanisms related to bortezomib treatment., (Copyright (c) 2009 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.)

The binding selectivity of charged liposomes to the spinal cord of rats affected by experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, was investigated. Positively and negatively charged liposomes were injected into the tail vein of rats, and blood/brain barrier (BBB) targeting was determined by confocal microscopy as a function of the temporal evolution of the inflammatory response. Accumulation in spinal cord endoneural vessels was observed for cationic, but not for anionic, liposomes, and only in EAE but not in healthy rats. The overall binding efficacy paralleled the severity of the clinical score, but targeting was observed already before clinical manifestation of inflammation. Preferential binding of positively charged liposomes in the course of acute EAE can be ascribed to subtle changes of BBB morphology and charge distribution in a similar way as for the binding of cationic particles to proliferating vasculature in chronic inflammation and angiogenesis. Our findings suggest that vascular changes related to increased binding affinity for cationic particles are very early events within the inflammatory reaction in acute EAE. Investigation of cationic vascular targeting can help to shed further light on these occurrences, and, potentially, new diagnostic and therapeutic options may become available. In neuroinflammatory diseases, cationic colloidal carrier particles may enable intervention at affected BBB by an approach which is independent from permeability increase.

Unlabelled: Diabetic peripheral neuropathy (DPN) can induce loss of nociception as well as mechanical hyperalgesia and tactile allodynia. Pharmacological and clinical studies have shown that buprenorphine, a low-molecular-weight, lipophilic, opioid analgesic available as a transdermal matrix patch formulation, acts on neuropathic pain. To assess the role of buprenorphine in the treatment of DPN-associated neuropathic pain, we used a well-established experimental rat model of DPN in which buprenorphine at doses of 1.2 and 2.4 microg/kg/h was administered by implantable Alzet osmotic pumps for 3 weeks. After 6 weeks of diabetes, nerve conduction velocity (NCV) and behavioural responses to noxious mechanical and thermal stimuli were assessed. Diabetic rats showed an impairment of NCV, mechanical allodynia, and thermal hypoalgesia. Both doses of buprenorphine significantly reversed the diabetes-induced allodynia up to day 7 of treatment. Buprenorphine did not alter either thermal perception or NCV., Perspective: This study evaluated, through a multimodal approach, the analgesic effect of buprenorphine in an experimental rat model of painful DPN. Our results suggest a possible role for buprenorphine in the management of DPN-associated neuropathic pain.

Epothilones are a novel class of microtubule-targeting anticancer agents that are neurotoxic. In this study, we investigated the epothilone B toxic effect in vitro and we characterized in vivo the general and neurological side effects of epothilone B administration in Wistar and Fischer rats. The in vitro experiments made it possible to explore a wide concentration range (0.1 nM-1 muM) and evidenced a dose-dependent effect of epothilone B exposure on neuron neurite elongation. This dose-dependent neurotoxic effect was confirmed in both in vivo studies performed on two different rat strains at the neurophysiological, behavioral and pathological levels in the dose range 0.25-1.5 mg/kg iv weekly x 4 weeks and tubulin hyper-polymerization was demonstrated in sciatic nerve specimens. These are the first studies of the neurological effects of epothilone B and they can provide a basis for extending pre-clinical investigation to other members of the epothilone family.

We have characterised for the first time the general and neurological side effects experienced when using a series of chronic non-lethal cisplatin + paclitaxel schedules in Wistar rats, selected according to our previous experience and the animals' maximum tolerated dose. At the pathological level, the use of combination schedules was definitely more toxic at the kidney and sternal bone marrow level than the single-agent schedules. At the neurophysiological examination based on the assessment of the nerve conduction velocity measurement in the tail nerve, we identified only one combination schedule that was more neurotoxic than the similar schedules based on single-agent administration. This observation was confirmed by the neuropathological examination performed on the sciatic nerve, dorsal root ganglia, ventral and dorsal roots. Our study supports the hypothesis that the general and, to a lesser extent, neurological effects of a combination of cisplatin and paclitaxel are different from those of the administration of both drugs as single agents. We believe that these models may be useful for testing neuroprotective strategies.

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.

Actively induced Lewis rat Experimental Autoimmune Encephalomyelitis (EAE) is a highly reproducible model for portraying the acute phase of multiple sclerosis. Our aim was to get more information about this model by means of flow cytometry looking at potential markers for tracing new treatments' efficacy. Thus we characterized the changes occurring in encephalitogenic TCR Vbeta8.2(+) frequency and the adhesion molecule alpha4 integrin expression in both spleen and spinal cord T cells. The increase in both these parameters was observed only in spinal cord infiltrating T cells while relevant changes in spleen cell composition were observed as early as disease onset.

Background: Antineoplastic drugs, such as cisplatin (CDDP), induce disabling peripheral neuropathies, representing a hindrance to effective cancer treatments. The exact pathogenesis of CDDP-induced neuropathy is not yet understood, and the dysregulation of gene expression has been proposed. Valproate (VPA) is an antiepileptic drug recently discovered to remodel gene expression, with hypothetically putative neuroprotective effects., Materials and Methods: VPA was tested in both, in vitro and in vivo models of CDDP-neurotoxicity., Results: VPA administered in combination with CDDP promoted dorsal root ganglia (DRG) neurons survival. Moreover, this treatment induced in Wistar rats an improvement of body weight, sensory nerve conduction velocity, and DRG morphometric analysis. In contrast, VPA was not able to rescue CDDP pre-treated rats., Conclusion: When used in combination with CDDP, VPA displays a protective action against neuropathy, in our models, suggesting possible future clinical applications.

Anthracyclines and anthracenediones are important oncotherapeutics; however, their use is associated with irreversible and cumulative cardiotoxicity. A novel aza-anthracenedione, pixantrone (BBR 2778), was developed to reduce treatment-related cardiotoxicity while retaining efficacy. This study evaluates the cumulative cardiotoxic potential of pixantrone compared with equiactive doses of doxorubicin and mitoxantrone in both doxorubicin-pretreated and doxorubicin-naïve mice. CD1 female mice were given doxorubicin 7.5 mg/kg (once weekly for 3 weeks) followed 6 weeks later by either sterile 0.9% saline, doxorubicin 7.5 mg/kg, pixantrone 27 mg/kg, or mitoxantrone 3 mg/kg (once weekly for 3 weeks). A second group of CD1 female mice were given 2 cycles of either sterile 0.9% saline, pixantrone 27 mg/kg, doxorubicin 7.5 mg/kg, or mitoxantrone 3 mg/kg (once weekly for 3 weeks). Animals were sacrificed at different time points for histopathologic examination of the heart. In the doxorubicin-pretreated mice, further exposure to doxorubicin or mitoxantrone resulted in a significant worsening of pre-existing degenerative cardiomyopathy. In contrast, pixantrone did not worsen pre-existing cardiomyopathy in these animals. Only minimal cardiac changes were observed in mice given repeated cycles of pixantrone, while 2 cycles of doxorubicin or mitoxantrone resulted in marked or severe degenerative cardiomyopathy. These animal studies demonstrate the reduced cardiotoxic potential of pixantrone compared with doxorubicin and mitoxantrone. Exposure to pixantrone did not worsen pre-existing cardiomyopathy in doxorubicin-pretreated mice, suggesting that pixantrone may be useful in patients pretreated with anthracyclines.

Bortezomib is a new proteasome inhibitor with a high antitumor activity, but also with a potentially severe peripheral neurotoxicity. To establish a preclinical model and to characterize the changes induced on the peripheral nerves, dorsal root ganglia (DRG) and spinal cord, bortezomib was administered to Wistar rats (0.08, 0.15, 0.20, 0.30 mg/kg/day twice [2q7d] or three times [3q7d] weekly for a total of 4 weeks). At baseline, on days 14, 21 and 28 after the beginning the treatment period and during a 4-week follow-up period sensory nerve conduction velocity (SNCV) was determined in the tail of each animal. Sciatic nerve, DRG and spinal cord specimens were processed for light and electron microscope observations and morphometry. At the maximum tolerated dose bortezomib induced a significant reduction in SNCV, with a complete recovery at the end of the follow-up period. Sciatic nerve examination and morphometric determinations demonstrated mild to moderate pathological changes, involving predominantly the Schwann cells and myelin, although axonal degeneration was also observed. Bortezomib-induced changes were also observed in DRG and they were represented by satellite cell intracytoplasmatic vacuolization due to mitochondrial and endoplasmic reticulum damage, closely resembling the changes observed in sciatic nerve Schwann cells. Only rarely did the cytoplasm of DRG neurons has a dark appearance and clear vacuoles occurring in the cytoplasm. Spinal cord was morphologically normal. This model is relevant to the neuropathy induced by bortezomib in the treatment of human malignancies and it could be useful in increasing our knowledge regarding the mechanisms underlying bortezomib neurotoxicity.

This study examined the dose-dependent efficacy of erythropoietin (EPO) for preventing and/or treating cisplatin (CDDP) induced peripheral neurotoxicity (CINP), and its influence on tumour treatment and growth. Rats received eight intraperitoneal (ip) injections of 2 mg/kg CDDP twice weekly. EPO co-administered (50 or 10 microg/kg ip, three times/week) had a dose-dependent effect, partially preventing CINP, but 0.5 microg/kg ip was not effective. The neuroprotective effect lasted at least 5 weeks after the last dose of EPO and CDDP. In addition, EPO (50 microg/kg ip three times/week) after the last injection of CDDP still induced a significant recovery of CINP. In a separate experiment in rats bearing mammary carcinoma EPO treatment (50 microg/kg ip) given concurrently with CDDP (1.0 and 1.5 mg/kg twice a week for four weeks) was neuroprotective without influencing the effectiveness of the treatment or tumour growth. EPO thus appears to be an effective neuroprotectant that does not interfere with tumour treatment.

Purpose: Antineoplastic drugs, such as cisplatin (CDDP), are severely neurotoxic, causing disabling peripheral neuropathies with clinical signs known as chemotherapy-induced peripheral neurotoxicity. Cotreatment with neuroprotective agents and CDDP has been proposed for preventing or reversing the neuropathy. Erythropoietin given systemically has a wide range of neuroprotective actions in animal models of central and peripheral nervous system damage. However, the erythropoietic action is a potential cause of side effects if erythropoietin is used for neuroprotection. We have successfully identified derivatives of erythropoietin, including carbamylated erythropoietin, which do not raise the hematocrit but retain the neuroprotective action exerted by erythropoietin., Experimental Design: We have developed previously an experimental chemotherapy-induced peripheral neurotoxicity that closely resembles CDDP neurotoxicity in humans. The present study compared the effects of erythropoietin and carbamylated erythropoietin (50 microg/kg/d thrice weekly) on CDDP (2 mg/kg/d i.p. twice weekly for 4 weeks) neurotoxicity in vivo., Results: CDDP given to Wistar rats significantly lowered their growth rate (P < 0.05), with slower sensory nerve conduction velocity (P < 0.001) and reduced intraepidermal nerve fibers density (P < 0.001 versus controls). Coadministration of CDDP and erythropoietin or carbamylated erythropoietin partially but significantly prevented the sensory nerve conduction velocity reduction. Both molecules preserved intraepidermal nerve fiber density, thus confirming their neuroprotective effect at the pathologic level. The protective effects were not associated with any difference in platinum concentration in dorsal root ganglia, sciatic nerve, or kidney specimens., Conclusions: These results widen the spectrum of possible use of erythropoietin and carbamylated erythropoietin as neuroprotectant drugs, strongly supporting their effectiveness.

Pixantrone is an immunesuppressor similar to mitoxantrone but with lower cardiotoxicity. We evaluated the effect of pixantrone on B cells and lymphomononuclear cells in the course of acute EAE. Pixantrone reduced the number of B cells and suppressed myelin basic protein (MBP) specific IgG production. In vitro, pixantrone induced apoptosis of rat B lymphocytes in a way similar to mitoxantrone. In addition, pixantrone inhibited antigen specific and mitogen induced lymphomononuclear cell proliferation, as well as IFN-gamma production, during EAE. These findings suggest a similar mechanism of action for pixantrone and mitoxantrone on the effector function of lymphomonocyte B and T cells.

Erythropoietin (EPO) possesses generalized neuroprotective and neurotrophic actions. We tested the efficacy of recombinant human EPO (rhEPO) in preventing and reversing nerve dysfunction in streptozotocin (STZ)-induced diabetes in rats. Two days after STZ [60 mg/kg of body weight (b.w.), i.p.], diabetic animals were administered rhEPO (40 microg/kg of b.w.) three times weekly for 5 weeks either immediately (preventive) before or after a 5-week delay (therapeutic) after induction of hyperglycemia or at a lower dose (8 microg/kg of b.w. once per week) for 8 weeks (prolonged). Tail-nerve conduction velocities (NCV) was assessed at 5 and 11 weeks for the preventive and therapeutic schedule, respectively. Compared to nondiabetic rats, NCV was 20% lower after 5 weeks in the STZ group, and this decrease was attenuated 50% by rhEPO. Furthermore, the reduction of Na(+),K(+)-ATPase activity of diabetic nerves (by 55%) was limited to 24% in the rhEPO-treated group. In the therapeutic schedule, NCV was reduced by 50% after 11 weeks but by only 23% in the rhEPO-treated group. rhEPO treatment attenuated the decrease in compound muscle action potential in diabetic rats. In addition, rhEPO treatment was associated with a preservation of footpad cutaneous innervation, as assessed by protein gene product 9.5 immunostaining. Diabetic rats developed alterations in mechanical and thermal nociception, which were partially reversed by rhEPO given either in a preventative or therapeutic manner. These observations suggest that administration of rhEPO or its analogues may be useful in the treatment of diabetic neuropathy.

Extra corporeal photochemotherapy (ECP) is an immunomodulating procedure used in several nonneurological diseases which, similarly to multiple sclerosis, are likely to be due to T-cell-mediated autoimmunity and it is probable that ECP can modulate the normal activity of peripheral blood mononuclear cells (PBMC). Using the Lewis rat experimental allergic encephalomyelitis (EAE) model of human multiple sclerosis (MS) we examined the effect of extracorporeal UV-A irradiation on psoralen-activated PBMC. In our experiment the comparison between the two groups of animals (ECP or sham-treatment) evidenced that the ECP treatment reduced the severity of EAE on clinical grounds and this result was confirmed by the pathological examination. The changes in the titers of anti-myelin antigen antibodies typical of EAE were also modulated by the procedure. Ex vivo examination evidenced a significant reduction in tumor-necrosis factor-alpha (TNF-alpha) released by PBMC after lipopolysaccharides (LPS) stimulation in culture. We conclude that ECP modifies the normal activity of PBMC during the course of EAE and it is possible that one of the anti-inflammatory mechanisms of action of ECP is correlated to a down-regulation of T-helper 1 lymphocytes activity.