A large-field polarisation-resolved laser scanning microscope: applications to CARS imaging.

Laser-scanning imaging techniques are frequently used to probe the molecule spatial orientation in a sample of interest by exploiting selection rules depending on the polarisation of the excitation light. For the successful implementation of these techniques the precise control of the polarisation at the sample level is of fundamental importance. Polarisation distortions induced by the optical elements are often the main limitation factor for the maximum size of the field-of-view in polarisation-resolved (PR) laser-scanning microscopy, since for large scanning angles the polarisation distortions may mask the real sample structure. Here we shall demonstrate the implementation of large-field-of-view PR microscopy and show PR CARS imaging of mouse spinal cord thanks to a careful design of the laser-beam optical path. We shall show that this design leads to strongly suppressed distortions and quantify their effects on the final images. Although the focus of this work is on CARS imaging, we stress that the approaches described here can be successfully applied to a wide range of PR laser-scanning techniques.

Bortezomib-induced peripheral neurotoxicity in human multiple myeloma-bearing mice.

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.

Bortezomib treatment produces nocifensive behavior and changes in the expression of TRPV1, CGRP, and substance P in the rat DRG, spinal cord, and sciatic nerve.

To investigate neurochemical changes associated with bortezomib-induced painful peripheral neuropathy (PN), we examined the effects of a single-dose intravenous administration of bortezomib and a well-established "chronic" schedule in a rat model of bortezomib-induced PN. The TRPV1 channel and sensory neuropeptides CGRP and substance P (SP) were studied in L4-L5 dorsal root ganglia (DRGs), spinal cord, and sciatic nerve. Behavioral measures, performed at the end of the chronic bortezomib treatment, confirmed a reduction of mechanical nociceptive threshold, whereas no difference occurred in thermal withdrawal latency. Western blot analysis showed a relative increase of TRPV1 in DRG and spinal cord after both acute and chronic bortezomib administration. Reverse transcriptase-polymerase chain reaction revealed a decrease of TRPV1 and CGRP mRNA relative levels after chronic treatment. Immunohistochemistry showed that in the DRGs, TRPV1-, CGRP-, and SP-immunoreactive neurons were mostly small- and medium-sized and the proportion of TRPV1- and CGRP-labeled neurons increased after treatment. A bortezomib-induced increase in density of TRPV1- and CGRP-immunoreactive innervation in the dorsal horn was also observed. Our findings show that bortezomib-treatment selectively affects subsets of DRG neurons likely involved in the processing of nociceptive stimuli and that neurochemical changes may contribute to development and persistence of pain in bortezomib-induced PN.

Murine neural stem cells model Hunter disease in vitro: glial cell-mediated neurodegeneration as a possible mechanism involved.

Mucopolysaccharidosis type II (MPSII or Hunter Syndrome) is a lysosomal storage disorder caused by the deficit of iduronate 2-sulfatase (IDS) activity and characterized by progressive systemic and neurological impairment. As the early mechanisms leading to neuronal degeneration remain elusive, we chose to examine the properties of neural stem cells (NSCs) isolated from an animal model of the disease in order to evaluate whether their neurogenic potential could be used to recapitulate the early phases of neurogenesis in the brain of Hunter disease patients. Experiments here reported show that NSCs derived from the subventricular zone (SVZ) of early symptomatic IDS-knockout (IDS-ko) mouse retained self-renewal capacity in vitro, but differentiated earlier than wild-type (wt) cells, displaying an evident lysosomal aggregation in oligodendroglial and astroglial cells. Consistently, the SVZ of IDS-ko mice appeared similar to the wt SVZ, whereas the cortex and striatum presented a disorganized neuronal pattern together with a significant increase of glial apoptotic cells, suggesting that glial degeneration likely precedes neuronal demise. Interestingly, a very similar pattern was observed in the brain cortex of a Hunter patient. These observations both in vitro, in our model, and in vivo suggest that IDS deficit seems to affect the late phases of neurogenesis and/or the survival of mature cells rather than NSC self-renewal. In particular, platelet-derived growth factor receptor-α-positive (PDGFR-α+) glial progenitors appeared reduced in both the IDS-ko NSCs and in the IDS-ko mouse and human Hunter brains, compared with the respective healthy controls. Treatment of mutant NSCs with IDS or PDGF throughout differentiation was able to increase the number of PDGFR-α+ cells and to reduce that of apoptotic cells to levels comparable to wt. This evidence supports IDS-ko NSCs as a reliable in vitro model of the disease, and suggests the rescue of PDGFR-α+ glial cells as a therapeutic strategy to prevent neuronal degeneration.

The glutamatergic neurotransmission in the central nervous system.

Glutamate is one of the major neurotrasmitters in mammalian brain and changes in its concentration have been associated with a number of neurological disorders, including neurodegenerative, cerebrovascular diseases and epilepsy. Moreover, recently a possible role for glutamatergic system dysfunction has been suggested also in the peripheral nervous system. This chapter will revise the current knowledge in the distribution of glutamate and of its receptors and transporters in the central nervous system.

Tissue distribution of glutamate carboxypeptidase II (GCPII) with a focus on the central and peripheral nervous system.

Glutamate carboxypeptidase II, also known as prostate specific membrane antigen or folate hydrolase I, is a type II transmembrane 750 amino acid membrane-bound glycoprotein, with a molecular weight in the human form of approximately 100 kDa and a demonstrated metallopeptidase activity. At the synaptic level it hydrolyzes N-acetylaspartylglutamate to N-acetyl-aspartate and glutamate. Its localization in the animal and human nervous system has only recently been clearly established, since many of the older studies gave conflicting results, likely due to the use of poorly characterized antibodies lacking epitope mapping and proper controls (i.e. immunohistochemistry complemented by western blot analysis and enzyme activity determination). In this chapter, we will review the available literature describing the animal and human distribution of glutamate carboxypeptidase in the central and peripheral nervous system.

Expression, distribution and glutamate uptake activity of high affinity-excitatory aminoacid transporters in in vitro cultures of embryonic rat dorsal root ganglia.

Glutamate is the major mediator of excitatory signalling in the mammalian central nervous system, but it has recently been shown to play a role in the transduction of sensory input at the periphery and in peripheral neuropathies. New advances in research have demonstrated that rat peripheral sensory terminals and dorsal root ganglia (DRG) express molecules involved in glutamate signalling, including high-affinity membrane-bound glutamate transporters (GLAST [glutamate aspartate transporter], GLT1 [glutamate transporter 1], EAAC1 [excitatory aminoacid transporter 1]) and that alterations in their expression and/or functionality can be implicated in several models of peripheral neuropathy, neuropathic pain and hyperalgesia. Here we describe, through immunoblotting, immunofluorescence assays and ß-counter analysis of [(3)H] l-glutamate uptake, the expression, distribution and activity of the glutamate transporters in in vitro cultures of embryonic dorsal root ganglia sensory neurons, sensory neurons+satellite cells and satellite cells. In this work we demonstrated that glutamate transporters are expressed in all cultures with a peculiar pattern of distribution. Even if GLAST is strongly detected in satellite cells, it is slightly expressed also in sensory neurons. GLT1 immunostaining is very weak in DRG neurons, but it was evident in the satellite cells. Finally, EAAC1 is localized in the soma and in the neuritis of sensory neurons, while it is not detectable in satellite cells. Moreover, all the cell cultures showed a strong sodium-energy-dependent glutamate uptake activity and it is more marked in neurons alone or in co-culture with satellite cells compared to satellite cells alone. Finally, we show that the complete or partial pharmacological inhibition of glutamate transporters virtually completely or partially abolish glutamate uptake in all cell culture. These results, that demonstrate that functionally active glutamate transporters can be studied in dorsal root ganglia cell cultures, provide further evidence for a role of glutamatergic transport in the peripheral nervous system and will be useful for testing whether any changes occur in in vitro models of peripheral nervous system damage.

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.

The role of oxidative stress and anti-oxidant treatment in platinum-induced peripheral neurotoxicity.

Platinum-based anticancer drugs are a cornerstone of the current antineoplastic treatment. However, their use is limited by the onset of peripheral nervous system dysfunction, which can be severe and persistent over a long period of time. Among the several hypothesis proposed to explain this side effect, evidence is increasing that dorsal root ganglia (DRG) oxidative stress can be an important pathogenetic mechanism and, possibly, a therapeutic target to limit the severity of platinum-induced peripheral neurotoxicity but preserving the anticancer effectiveness. In fact, DRG energy failure has been suggested as a result of mitochondrial DNA-platinum binding and several antioxidant drugs have been tested in pre-clinical experiments and clinical trials. In this review, an update on the current knowledge on the relationship existing between oxidative stress and platinum drugs peripheral neurotoxicity will be given.

Neuropathological changes in the peripheral nervous system and spinal cord in a transgenic mouse model of Niemann-Pick disease type A.

OBJECTIVE: Type A Niemann-Pick is a severe neurological disease, caused by a mutation of the gene of acid sphingomyelinase (ASM) and reduced enzyme activity. Some studies reported neuropathological changes occurring in the central nervous system of ASM deficient transgenic (ASMKO) mice, while a detailed study on the peripheral nervous system (PNS) at different ages is currently lacking. The aim of our study was to examine the pathological changes occurring in the PNS and in the spinal cord in an AMSKO model of Niemann-Pick disease (NPD) Type A. MATERIAL AND METHOD: Dorsal root ganglia (DRG), peripheral nerves and spinal cord specimens were obtained from ASMKO mice and age-matched wild type animals (age range = 1-7 months). They were observed at the light and electron microscope. Behavioral testing was performed to assess motor coordination and reactivity. Fluoro-Jade B was used as a high affinity fluorescent marker for degenerating neurons. RESULTS: Typical NPD cytoplasmic inclusions were observed in DRG neurons and satellite cells, in peripheral nerve Schwann cells, in spinal cord neurons and in endothelial cells. All these inclusions were present from the age of 1 month and increased with aging. By Fluoro-Jade B staining we demonstrated the occurrence of neuronal degeneration starting from 5 months of age. CONCLUSION: Despite the fact that a definite diagnosis of NPD Type A depends on enzymatic assays and/or molecular analysis, morphological investigation remains an important diagnostic procedure. Well-defined and complete neuropathological information about the ASMKO mouse model, inclusive of PNS examination, may be crucial in the pre-clinical evaluation of new therapies.

Proteasome inhibition: a promising strategy for treating cancer, but what about neurotoxicity?

The inhibition of protein degradation through the ubiquitin-proteasome pathway is a recently developed approach to cancer treatment which extends the range of cellular targets for chemotherapy. This therapeutic strategy is very interesting since the proteasomes carry out the regulated degradation of unnecessary or damaged cellular proteins, a process that is dysregulated in many cancer cells. Based on this hypothesis, the proteasome complex inhibitor bortezomib was approved for use in multiple myeloma patients by the US Food and Drug Administration (FDA) in 2003 and by the European Medicines Agency (EMEA) in 2004, and several new drugs with the same target and, sometimes, mechanism of action are currently under development. Interestingly, proteasome inhibitors have now also been tested in combination chemotherapy for the treatment of several solid tumors and it is likely that there will be more generalized use of these compounds in the near future. Despite its remarkable effectiveness, which led to it being rapidly approved for clinical use, some concern has been raised regarding the safety of bortezomib (and in general of proteasome inhibitors) since reduced degradation of damaged proteins has been postulated as being the basic mechanism of severe neurological diseases affecting the central nervous system. While this concern has not been confirmed by the clinical course of treated patients, from the first Phase I studies, it emerged that peripheral sensory neurotoxicity was one of the major dose-limiting toxicities. The main results from the use of proteasome inhibition in cancer chemotherapy and the implications for treatment on the nervous system will be reviewed.

Extracorporeal photochemotherapy reduces the severity of Lewis rat experimental allergic encephalomyelitis through a modulation of the function of peripheral blood mononuclear cells.

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.

Cisplatin-induced peripheral neurotoxicity in rats reduces the circulating levels of nerve growth factor.

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.

Circulating nerve growth factor level changes during oxaliplatin treatment-induced neurotoxicity in the rat.

BACKGROUND: Oxaliplatin neurotoxicity represents a clinically-relevant problem and its etio-pathogenesis is still unknown. We explored the possible role of some neuronal growth factors ("neurotrophins") during the course of oxaliplatin sensory neuronopathy. MATERIALS AND METHODS: In our rat model two different doses of oxaliplatin were used (2 and 3 mg/kg i.v. twice weekly for 9 times). The neurotoxicity of the treatment was assessed with neurophysiological and pathological methods and serum neurotrophin levels were measured by ELISA. RESULTS: Both oxaliplatin-treated groups showed the neurophysiological and neuropathological changes which mimic the chronic effects of oxaliplatin administration in humans, e.g. reversible sensory impairment due to dorsal root ganglia neuron damage. These changes were associated with a significant and dose-dependent reduction only in the circulating level of nerve growth factor (NGF), which returned to normal values after neurophysiological and pathological recovery. CONCLUSION: This specific association between neurological impairment and NGF modulation indicates that NGF impairment has a role in the neurotoxicity of oxaliplatin.

Effects of different schedules of oxaliplatin treatment on the peripheral nervous system of the rat.

The aim of this study was to determine the influence of oxaliplatin scheduling on the onset of peripheral neurotoxicity and ototoxicity in a rat model. Animals were treated with four different schedules of oxaliplatin using two cumulative doses (36 and 48 mg/kg intraperitoneally (i.p.)). The neuropathological examination evidenced dorsal root ganglia (DRG) nucleolar, nuclear and somatic size reduction with nucleolar segregation in the treated rats. Sensory nerve conduction velocity (SNCV) was reduced after oxaliplatin treatment, while the auditory pathway was unaffected. After treatment, platinum was detected in the kidney, DRG and sciatic nerve. After a 5-week follow-up period, recovery of the pathological changes in the DRG and sciatic nerves occurred, although platinum was still detectable in these tissues. The following conclusions may be drawn: the main targets of oxaliplatin neurotoxicity were the DRG; the shorter the interval between the injections, the higher the severity of peripheral neuropathy and this was also related to the cumulative oxaliplatin dose; the peripheral neurotoxicity tended to be reversible; ototoxicity was absent even with high cumulative doses of oxaliplatin.

Lipid-free versus lipid-bound P2 protein-induced experimental allergic neuritis: clinicopathological, neurophysiological, and immunological study.

The P2 protein of the peripheral nervous system myelin is a neuritogenic protein capable of inducing experimental allergic neuritis (EAN) in the Lewis rat. It has been suggested that the addition of some lipids to the protein isolated in the lipid-free form might enhance its immunogenicity. In this study, we compared lipid-free P2 (the EAN factor) and the corresponding lipid-bound form of the protein regarding their ability to induce EAN. Lipid-bound P2, copurified with all the myelin lipids, shows a conformation different from that of LF-P2. The timing of disease and the clinical scores of lipid-bound P2-induced EAN animals (n = 23) did not differ statistically from those injected with lipid-free P2 (n = 23), with only a tendency to higher clinical severity in the former group. Tail nerve conduction velocities did not differ in the two groups and in both were significantly lower in comparison to Freund adjuvant controls (n = 8). Inflammation and demyelination predominated in the spinal roots and were less evident in the sciatic nerve for both groups of animals. The ELISA determination of antibodies to lipid-free and lipid-bound P2 revealed the development of antibodies recognizing the lipid-free form of the protein in both groups of animals. Our results stand in contrast to results of previous studies performed after addition of exogenous lipids to the P2 purified in the lipid-free form and indicate that lipid-bound P2 is not significantly more immunogenic than lipid-depleted P2.

Anti-sulfatide IgM antibodies in peripheral neuropathy.

Anti-sulfatide IgM antibodies have been recently associated with neuropathy but the clinical and electrophysiological correlations of this reactivity remains unclear. We reviewed the clinical and electrophysiological features of patients with high anti-sulfatide titers detected in our laboratory from 1991 to 1998. Of the 564 patients with different neurological diagnosis tested by enzyme-linked immunosorbent assay (ELISA), 11 had high anti-sulfatide IgM titers (>1/8000), 26 had titers of 1/8000 while 78 had titers of 1/4000. All patients with high anti-sulfatide IgM titers had a chronic, dysimmune, mostly sensorimotor neuropathy that in seven was associated with IgM monoclonal gammopathy. In most of these patients electrophysiological and morphological studies were consistent with a predominantly demyelinating neuropathy frequently associated with prominent axonal loss. Antibody titers of 1/8000, though always associated with neuropathy, did not correlate with a particular form or cause of neuropathy, while lower titers were equally distributed in patients with different neurological disorders. Our study indicate that high anti-sulfatide IgM titers (>1/8000) are highly predictive for a chronic, dysimmune, mostly demyelinating neuropathy often associated with IgM monoclonal gammopathy, and may therefore have potential diagnostic relevance.

Distribution of paclitaxel within the nervous system of the rat after repeated intravenous administration.

The distribution of paclitaxel (Taxol) within the central and peripheral nervous system after repeated administration of this antineoplastic agent is still largely unknown. In this study we determined for the first time paclitaxel tissue concentration in the brain, spinal cord, dorsal root ganglia (DRG) and sciatic nerve using an experimental paradigm in the rat which reproduces the features of paclitaxel peripheral neurotoxicity in humans. Pathological confirmation of the onset of paclitaxel-induced peripheral neurotoxicity was performed. In order to achieve reliable results even with low concentrations of paclitaxel, a newly reported analytical method (high-performance liquid chromatography with tandem mass spectrometry) was used. We demonstrated that paclitaxel has easy access to the DRG, where it accumulates, while the lowest concentrations of the drug were measured in the brain. The intermediate concentrations of paclitaxel observed in the sciatic nerve and spinal cord may be due to paclitaxel transport along the centrifugal and centripetal branches of the DRG neuron axons.

Effect on the peripheral nervous system of systemically administered dimethylsulfoxide in the rat: a neurophysiological and pathological study.

The issue of dimethylsulfoxide (DMSO) neurotoxicity is an important one, given its wide use in experimental toxicology as a solvent for hydrophobic substances. We examined the effect of the intraperitoneal administration of different DMSO solutions (1.8-7. 2%) on the peripheral nervous system of Wistar rats treated for 10 consecutive days and followed-up for an additional 45 days. DMSO administration induced a dose-dependent reduction in nerve conduction velocity, with complete recovery occurring in the follow-up. No structural changes were found in the sciatic nerve at 1.8% and 3.6% DMSO concentrations, suggesting that the mechanism of action of DMSO involves a functional impairment (i.e. conduction block) similar to that already described for this substance in isolated systems. However, when DMSO was administered at the 7.2% concentration, evident structural changes were observed in the sciatic nerve, with myelin disruption and uncompacted myelin lamelle. The neurophysiological and pathological changes observed in our study are severe enough to merit careful consideration in the course of experimental studies involving DMSO as a solvent for drugs which are under evaluation for their potential neurotoxicity.