Chemotherapy-induced peripheral neuropathy in immunodeficient mice: new useful ready-to-use animal models

Cisplatin, paclitaxel, and bortezomib represent the most employed chemotherapy regimens for the treatment of genitourinary cancers, breast and lung cancers and multiple myeloma. Nevertheless, their clinical use is often associated to the development of peripheral neuropathies characterized mostly by sensory alterations and pain (Argyriou et al., 2012). Several rat models of chemotherapy-induced peripheral neuropathy (CIPN) had been established in the past to describe the mechanisms of its development and pathogenesis. However, only few cancer cell lines induce the development of cancer in the rat, while immunodeficient mice best allowed human cancers xenografts to study at the same time, the antineoplastic and neurotoxic effects of chemotherapy. Here we characterized neuropathic pain, neurophysiological and neuropathological alterations induced by chronic chemotherapy in immunodeficient nude mice. Mice were treated with effective doses of cisplatin (4 mg/Kg, i.p), paclitaxel (80 mg/Kg, i.v) and bortezomib (0.8 mg/Kg, i.v) for a 4-6 weeks period. At the end of the 6th week all chemotherapy regimens determined a significant impairment of neurophysiologic parameters, mechanical allodynia and thermal hypo-or hyperalgesia. Light microscopy analysis of dorsal root ganglia (DRG) showed that bortezomib induced morphological alterations in the sensory neurons and satellite cells as dark inclusions and clear vacuolation throughout the cytoplasm. Moreover, sporadic episodes of neuronal degeneration were evident. DRG of cisplatin-treated animals showed severe neuronal atrophy. Moreover bortezomib induced moderate to severe axonal degeneration of the myelinated fibers in the sciatic nerves. More severe changes were induced by paclitaxel where also areas of fibers loss were frequently observed and rare pathological abnormalities were present in unmyelinated fibers. Similar changes were evident in paclitaxel-treated mice (degeneration at different stage of severity in myelinated fibers, enlargement of Schwann cells, fibers loss and dark inclusions in the unmyelinated fibers). These schedules demonstrated to be effective in mimicking clinical features of painful neuropathies and allows to combine the study of peripheral neurotoxicity of chemotherapy drugs to their anti-tumour activity against cancers of human origin

A new animal model of chemotherapy induced peripheral neurotoxicity: the immune-deficient mouse

Cisplatin, paclitaxel and bortezomib are anticancer drugs widely employed in the treatment of different solid tumours even though peripheral neurotoxicity represents a major limitation in their clinical use. During the last decades many rat and mouse models of chronic chemotherapyinduced peripheral neurotoxicity (CIPN) have been characterized from the clinical, pathological, neurophysiological and behavioural point of view. These models were based on immune-competent animals, however in preclinical oncology immune-deficient mice are mainly used. In this respect, the development of immune-deficient mice models could represent a basis for the concurrent investigation of both anticancer drug efficacy and neurotoxicity in animals implanted with human-derived cancer. Moreover, in the same model, neuroprotective effects and non-interference with anticancer activity could be better studied. In this study we established the feasibility of new immune-deficient murine models of peripheral neurotoxicity induced by three anticancer drugs. Forty-eight athymic nude mice were randomized in 4 groups of 12 animals, three were treated respectively with cisplatin, paclitaxel and bortezomib, and one was left untreated. All animals were followed up for 6 weeks. They were examined at baseline, week 4 and 6 for neurophysiological functions and behavioural tests, whilst morphological and morphometric analysis were performed on dorsal root ganglia (DRG) and peripheral nerves collected after 4 and 6 weeks of treatment. The results of the study demonstrate that athymic nude mice show CIPN features similar to those observed in conventional models even if some differences must be remarked as the prolonged time of treatment required to develop a chronic neuropathy. The characterization of this new mice model of CIPN will allow studies of antineoplastic and neurotoxic effects in the same animal

Oxaliplatin-induced peripheral neurotoxicity: morphological characterization in different mouse strains

Oxaliplatin is one of the most effective anticancer drug, particularly employed in the treatment of colorectal cancer, but one of the major limitation in its use is peripheral neurotoxicity. Oxaliplatin induced peripheral neurotoxicity (OIPN) has a high incidence and is frequently long lasting or permanent. Neuropathy is characterized by distal sensory impairment initially in the legs, then extending to the arms. A prominent manifestation of sensitive damage is ataxia. Besides chronic neurotoxicity, many patients experience an acute, rapidly developing cold-induced sensory neuropathy, usually resolving within one week. OIPN clinical manifestations reflect the involvement of dorsal root ganglia (DRG) as primary target of the drug toxicity. Although this assumption is largely accepted and some pathogenetic hypothesis have been proposed, mechanisms at the basis of OIPN need to be clearly defined. OIPN may vary in frequency and severity among different cancer patients despite equal treatment schedules. A genetic susceptibility for more severe oxaliplatin-induced peripheral neurotoxicity (OIPN) has been suggested but never confirmed. Therefore we designed a study to assess the phenotypic differences induced by oxaliplatin treatment in six different mice strains (Balb c, AJ, C57Bl6, FVB, DBA, CD1) aiming at identifying the more and less severely affected. Animals were treated with OHP 3.5 mg/Kg/iv twice weekly x 4 weeks and evaluated before and after treatment. In all strains we performed a multimodal characterization of its neurotoxicity through morphological and morphometrical assessment in caudal nerve and DRG at light and electron microscopy, intra-epidermal nerve fibers density quantification, evaluation of mechanical and cold allodynia/hypoaesteshesia, caudal and digital nerve conduction velocity, activity of wide dynamic range (WDR) neurons of the spinal dorsal horn. Our preliminary data suggest that all the strains show signs of OIPN but not the same modifications in the parameters examined. We will show these results with particular attention to morphological data. This study suggests that genetic variability might have a role in the type and severity of OHP-induced peripheral damage

Stem cell augmented mesh materials: an in vitro and in vivo study

INTRODUCTION AND HYPOTHESIS: To test in vitro and in vivo the capability of mesh materials to act as scaffolds for rat-derived mesenchymal stem cells (rMSCs) and to compare inflammatory response and collagen characteristics of implant materials, either seeded or not with rMSCs. METHODS: rMSCs isolated from rat bone marrow were seeded and cultured in vitro on four different implant materials. Implants showing the best rMSC proliferation rate were selected for the in vivo experiment. Forty-eight adult female Sprague-Dawley rats were randomly divided into two treatment groups. The implant of interest-either seeded or not with rMSCs-was laid and fixed over the muscular abdominal wall. Main outcome measures were: in vitro, proliferation of rMSCs on selected materials; in vivo, the occurrence of topical complications, the evaluation of systemic and local inflammatory response and examination of the biomechanical properties of explants. RESULTS: Surgisis and Pelvitex displayed the best cell growth in vitro. At 90 days in the rat model, rMSCs were related to a lower count of neutrophil cells for Pelvitex and a greater organisation and collagen amount for Surgisis. At 7 days Surgisis samples seeded with rMSCs displayed higher breaking force and stiffness. CONCLUSIONS: The presence of rMSCs reduced the systemic inflammatory response on synthetic implants and improved collagen characteristics at the interface between biological grafts and native tissues. rMSCs enhanced the stripping force on biological explants

Age-related changes in the function and structure of the peripheral sensory pathway in mice

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\ua0months 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\ua0months, 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

APOA-1Milano muteins, orally delivered via genetically modified rice, show anti-atherogenic and anti-inflammatory properties in vitro and in Apoe-/- atherosclerotic mice

BACKGROUND: Atherosclerosis is a slowly progressing, chronic multifactorial disease characterized by the accumulation of lipids, inflammatory cells, and fibrous tissue that drives to the formation of asymmetric focal thickenings in the tunica intima of large and mid-sized arteries. Despite the high therapeutic potential of ApoA-1 proteins, the purification and delivery into the disordered organisms of these drugs is still limited by low efficiency in these processes. METHODS AND RESULTS: We report here a novel production and delivery system of anti-atherogenic APOA-1Milano muteins (APOA-1M) by means of genetically modified rice plants. APOA-1M, delivered as protein extracts from transgenic rice seeds, significantly reduced macrophage activation and foam cell formation in vitro in oxLDL-loaded THP-1 model. The APOA-1M delivery method and therapeutic efficacy was tested in healthy mice and in Apoe-/- mice fed with high cholesterol diet (Western Diet, WD). APOA-1M rice milk significantly reduced atherosclerotic plaque size and lipids composition in aortic sinus and aortic arch of WD-fed Apoe-/- mice as compared to wild type rice milk-treated, WD-fed Apoe-/- mice. APOA-1M rice milk also significantly reduced macrophage number in liver of WD-fed Apoe-/- mice as compared to WT rice milk treated mice. TRANSLATIONAL IMPACT: The delivery of therapeutic APOA-1M full length proteins via oral administration of rice seeds protein extracts (the 'rice milk') to the disordered organism, without any need of purification, might overcome the main APOA1-based therapies' limitations and improve the use of this molecules as therapeutic agents for cardiovascular patients

Innovative and Efficient Oral Delivery Method of APOA-1Milano Muteins Which Retain Anti-Atherosclerotic and Anti-Inflammatory Properties

Background. The management of modifiable risk factors exposure, in particular dyslipidemia, is the first line of intervention in preventing cardiovascular events. HDLs have been demonstrated to have anti-inflammatory and atheroprotective properties and, among HDLs, Apolipoprotein A-I (APOA-1), which promotes reverse cholesterol efflux, has been deeply investigated for the great therapeutic potential, particularly emphasized for the naturally occurring mutation APOA-1Milano (APOA-1M). Despite the high therapeutic potential of these molecules, their purification and delivery into the disordered organism is still limited by a very low efficiency in these processes. Aim. To develop an efficient system of production and delivery of APOA-1 muteins without need of purification. Methods and Results. Rice plants were genetically modified to express APOA-1M protein in their seeds. Protein extract from transgenic rice seeds (the ‘APOA-1M rice milk’) was tested for functionality in vitro on THP-1 macrophages exposed to oxLDL. Protein extract from wild type rice seeds (the ‘WT rice milk’) was used as control. The APOA-1M rice milk, but not the WT rice milk, significantly reduced expression of MCP-1 in oxLDL-loaded THP-1 macrophages in a dose-dependent manner and it promoted reverse cholesterol efflux in THP-1 macrophages. The lack of toxicity and the tolerability of the orally administered APOA-1M rice milk was evaluated in healthy mice. In an early-intermediate atherosclerotic mouse model (Apoe-/-mice fed with Western Diet for 8 weeks), 3 weeks of APOA-1M, but not the WT, rice milk treatment (15d, 5d/week, by oral gavage) significantly reduced area of lipid deposition and lipids concentration at aortic arch (en face analysis). Moreover, the APOA-1M, but not the WT, rice milk treatment reduced the hepatic CD68-positive cells and ameliorated the lipid management gene expression profile in liver of WD-fed Apoe-/-mice. Interestingly, all these findings were observed in mice still exposed to WD during the therapeutic treatment. Conclusion. The oral delivery of APOA-1M muteins, by means of genetically modified rice seeds extract, is athero-protective and anti-inflammatory even if the organism is exposed to high fat diet during the treatment, suggesting that this therapeutic approach could be effective in preventing and in counteracting atherogenic risk factors