Barbara Sala

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.

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.

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.

Evaluation of tubulin polymerization and chronic inhibition of proteasome as citotoxicity mechanisms in bortezomib-induced peripheral 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.

Islet transplantation and insulin administration relieve long-term complications and rescue the residual endogenous pancreatic β cells.

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.

Positive effect of Mesenchymal Stem Cells therapeutic administration on chronic Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a crippling chronic disease of the Central Nervous System caused by the presence of self-antibodies which progressively damage axonal myelin sheath, leading to axonal transmission impairment and to the development of neurological symptoms. MS is characterized by a Relapsing-Remitting course, and current therapies rely only on the use of immunosuppressive drugs, which are however unable to reverse disease progression. Encouraging results have been obtained in preclinical studies with the administration of Mesenchymal Stem Cells (MSCs) before disease onset (Zappia et al., 2005). Here, we investigate the therapeutic potential of MSC administration after disease onset into an animal model of MS, represented by Dark Agouti rats affected by chronic Relapsing-Remitting Experimental Autoimmune Encephalomyelitis (EAE) (Cavaletti et al., 2004). 106 MSC were intravenously injected in EAE rats after disease onset. Clinical score was assessed daily, and after 45 days rats were sacrificed and histological analysis of spinal cords performed to evaluate the demyelinating lesions. After the first peak of disease, no further relapses were observed in EAE rats treated with MSCs, differently from what observed in EAE group. Histological analysis demonstrated the presence of demyelinated plaques in spinal cords of EAE rats, (Luxol fast Blue staining and anti-MBP immunohystochemistry). On the contrary the therapeutic schedule with MSCs significantly reduces the number and the extension of demyelinated areas in the spinal cords, confirming clinical score evaluations. These results demonstrated that MSCs ameliorate the clinical course of EAE and hamper the disease relapsing by reducing the areas of demyelinated lesions. Granted by MIUR – FIRB Futuro in Ricerca 2008 Prot. N° RBFR08VSVI_001.

Influence of aging on peripheral nervous system: a morphological and morphometric study

It is well known that aging influences several functional and structural features of peripheral nerves (Verdu et al., 2000; Ceballos et al., 1999; Jeronimo et al., 2008). However, the role of these changes in the damage/repair mechanisms occurring in acquired peripheral neuropathies is still unclear. To this aim, a multimodal, long-term assessment in a mice model would represent an optimal tool to perform experimental neuropathy studies designed to evaluate the role of aging in relationship with a given nerve injury. In this study we used 40 females one-month-old C57B1/6 mice and we followed-up them for fifteen months. Digital and caudal nerve conduction velocity (NCV) studies were performed monthly to evaluate changes in electrophysiological features; moreover, four animals were sacrificed every two months in order to collect caudal nerve, sciatic nerve, dorsal root ganglia (DRG) and skin for morphological and morphometric analysis. The neurophysiological assessments showed a remarkable increase of caudal NCV until the age of 9 months and then it remained unchanged until the end of the observation period; in the same period, digital NCV increase was also present although less marked. At the pathological level, both caudal and sciatic nerves showed a decrease in fibres density related with age, whereas axon and fibres diameters tended to increase. These preliminary data can be considered a first step aiming at creating a background for future studies on the relationship between aging and peripheral nervous system induced damage.

Abstract 933: Peripheral neuropathy induced by chronic administration of Cisplatin, taxol and bortezomib in several murine models

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Cisplatin (CDDP), Taxol (TAX) and Bortezomib (BZ) are chemotherapeutic drugs commonly employed in clinical practice for the treatment of solid and haematological tumors. Their clinical use is limited by the development of a peripheral neuropathy characterized by sensory alterations, pain and, in part, by motor and autonomic dysfunctions. Several rat models had been performed to study and describe the mechanisms of the peripheral neurotoxicity induced by these drugs. However, since only few cancer cell lines are able to induce the development of cancer in the rat, we focused our attention on mice models to allow the combined study of the antineoplastic activity and of the neurotoxic effects of the anticancer compounds. Moreover, different mice strains should be useful for different kind of studies. Here we investigated the interactions between the mice genotype and their drug response to determine the susceptibility to the development of chemotherapy-induced peripheral neuropathy. To this aim, we investigated the neurophysiological and neuropathological alterations induced by the chronic treatment with different chemotherapy drugs in female Balb/c, CD1 and c57 mice and in male c57 and FVB mice. Mice were injected with Cisplatin (2, 4 mg/Kg, ip, 2qw), or Taxol (50, 70, 80 mg/Kg, iv, 1qw) or Bortezomib (0.4, 0.8 mg/Kg, iv, 2qw) for a period of 4-6 weeks. At the end of the treatment the nerve conduction velocities (NCVs) were determined in the caudal and in the digital nerves and the dorsal root ganglia (DRG) and sciatic nerves collected for the neuropathological analysis; behavioural tests were performed to study if these drugs are able to induce allodynia or hyperalgesia. In all mice models we observed that TAX and CDDP induced a significant alteration in body weight at the end of the treatment. All the drugs induced a significant reduction in the caudal and digital NCV in Balb/c and CD1 mice. TAX and BZ caused the axonal degeneration of the sciatic nerves while CDDP and BZ morphological alterations in the DRG. In c57 female mice, only BZ induced a significant reduction in caudal NCV while in c57 male mice only CDDP caused neurophysiological impairments. By contrast, male FVB mice showed any neurophysiological alterations. All these models, except FVB mice, have shown the onset of allodynia and hyperalgesia at the end of the treatment. In conclusion we can affirm that, even if at different extent, Balb/c, CD1 and c57 mice were able to develop the different chemotherapy-induced peripheral neuropathies. Moreover, also the gender seemed to be involved in the severity of the neuropathy. By contrast, FVB mice were less susceptible to the neurotoxic damage. This study would give a useful guideline for the choice of mouse strains in the combined studies of the antineoplastic activity and of the neurotoxic effects of chemotherapy drugs. Partially supported by a grant from “Fondazione Banca del Monte di Lombardia”. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 933. doi:1538-7445.AM2012-933

Abstract 5679: Characterization in vivo of two different molecular mechanisms involved in the development of bortezomib-induced peripheral neuropathy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Bortezomib (BTZ) is a chemotherapy drug with clinical efficacy in patients with relapsed/refractory, relapsed and newly diagnosed multiple mieloma (MM). It acts by inhibiting protein degradation by the proteasoma, but its clinical use is limited by a dose-limiting neurotoxicy and by the development of tumor resistance. BTZ-induced peripheral neuropathy may occur in cancer patients from a variety of mechanisms, among which proteasoma inhibition and the alteration of microtubules stability. To study in vivo these two different mechanisms involved in the onset of neuropathy, we used a well-characterized rat model of BTZ-induced peripheral neuropathy. Wistar rat were administered with BTZ 0.20 mg/kg, three times/week for eight weeks. At the end of the treatment period the relationship between neurotoxicity and the profile of the proteasoma inhibition was evaluated in different tissues. The level of 20S proteasoma inhibition was assessed by the proteasoma activity assay into blood mononuclear cells (PBMC), sciatic nerve and brain by fluorimetric assay at different time points. Moreover, we examined microtubule polymerization in sciatic nerve by comparing the distribution of acetylated tubulin (a post translational marker of stabilized microtubule) between polymerized (P) and soluble (S) fractions by western blot experiments. After eight weeks of treatment, we observed BTZ-related neurophysiologic alterations and neuropathological damages in the axons of sciatic nerves and in sensory neurons and satellite cells of dorsal root ganglia, demonstrating the onset of a peripheral neuropathy. The recovery of the proteasoma activity was observed within 24 hours from the drug administration if BTZ was injected in a single acute dose; while the proteasoma activity remained suppressed if the drug was chronically administered. This effect was probably due to a cumulative effect of chronic administration of BTZ on its biological target. Besides, in this study we increase in the amount of acetylated-tubulin in the polymerized fraction in BTZ-treated sciatic nerves as compared with control animals. This preclinical work provide a potential explanation for the development of BTZ-induced peripheral neuropathy, through the bortezomibs ability to induce a chronic proteasoma inhibition and to suppress the cytoskeleton dynamics. In conclusion, this model showed a toxic effect on peripheral nervous system induced by a prolonged BTZ-induced of proteasoma inhibition and stabilized microtubule. Therefore this model can be useful for the study of “de novo” proteasoma synthesis that can be useful for the recovery of its activity. Moreover this work will enable us to better characterize the potential role of microtubule stabilization in the neurotoxicity of bortezomib. Supported in part by grants from the “Fondazione Banca del Monte di Lombardia” Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5679. doi:1538-7445.AM2012-5679

Abstract 4388: LipoplatinTM: A less neurotoxic formulation of Cisplatin

Purpose: Cisplatin is one of the most effective cytotoxic agent in the treatment of solid malignancies, but its use is limited by several side effects. Among them, peripheral neurotoxicity can be dose-limiting. A liposomal formulation of cisplatin, LipoplatinTM, was developed to reduce the systemic toxicity of cisplatin but preventing its efficacy. Aim of this study was to test in an animal model through a multimodal approach if chronic treatment with two different schedules of LipoplatinTM selected in the range of its anticancer effective dose is less neurotoxic than cisplatin administrations. Methods: Female Wistar rats were treated intraperitoneally with cisplatin at the dose of 4mg/kg or with LipoplatinTM 12 and 24mg/kg once weekly for 4 times. General toxicity was assessed by daily observation, body weight change, hematological and blood chemistry analysis and histopathology of liver and kidney. The onset of the peripheral neurotoxicity was assessed using tail nerve conduction velocity (NCV), morphological and morphometrical analysis of dorsal root ganglia (DRG) and by morphological analysis of sciatic nerve. Results: Cisplatin induced a statistically significant reduction in body weight, the development of renal failure and the impairment in NCV with pathological alterations in the DRG and sciatic nerve. By contrast no significant weight gain reduction was observed in animals treated with both doses of LipoplatinTM. Moreover the lowest dose induced a less severe damage to the peripheral nervous system with a moderate decrease of NCV and mild pathological alterations of DRG and sciatic nerve. Conclusions: The results suggest that LipoplatinTM 12mg/kg is less neurotoxic than cisplatin 4mg/kg, thus opening the possibility to use this new formulation in future studies where its anticancer activity and the peripheral neurotoxicity will be assessed in parallel. This study was supported by “Fondazione Banca del Monte di Lombardia” Regulon supported this study exclusively by providing LipoplatinTM Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4388. doi:10.1158/1538-7445.AM2011-4388