Exposure to polystyrene nanoplastics induced physiological and behavioral effects on the brittle star Ophiactis virens.

Nanoplastic contamination has become an issue of environmental concern but the information on the potential adverse effects of nanoplastics on marine ecosystems is still limited. Therefore, the aim of this work was to investigate the effects of the exposure to polystyrene nanoplastics (PS-NPs; 0.05, 0.5 and 5 µg/mL) on the brittles star Ophiactis virens. Diverse endpoints at different levels of biological organization were considered, including behavior, arm regeneration capacity and oxidative stress. PS-NPs were observed on the brittle star body surface but not in inner tissues. Accumulation of PS-NPs was observed in the pre-buccal cavity of animals exposed to 5 µg/mL PS-NPs which also displayed delayed righting activity and an oxidative stress condition. Nevertheless, no effect was observed on arm regeneration efficiency at any tested PS-NPs concentration. Overall, our results highlighted that prolonged exposure to high amounts of PS-NPs could interfere at least partially with the physiology of O. virens.

The ß2V287L nicotinic subunit linked to sleep-related epilepsy differently affects fast-spiking and regular spiking somatostatin-expressing neurons in murine prefrontal cortex.

Mutant subunits of the neuronal nicotinic ACh receptor (nAChR) can cause Autosomal Dominant Sleep-related Hypermotor Epilepsy (ADSHE), characterized by frontal seizures during non-rapid eye movement (NREM) sleep. We studied the cellular bases of the pathogenesis in brain slices from mice conditionally expressing the ADSHE-linked ß2V287L nAChR subunit. ß2V287L mice displayed minor structural alterations, except for a ~10% decrease of prefrontal cortex thickness. However, they showed a substantial decrease of the excitatory input to layer V fast-spiking (FS) interneurons, despite a concomitant increase in the number of glutamatergic terminals around the cell soma. Hence, prefrontal hyperexcitability may depend on a permanent impairment of surround inhibition. The effect disappeared when ß2V287L was silenced until postnatal day 15th, suggesting that the transgene selectively affects the maturation of glutamatergic synapses on FS neurons. The other main population of interneurons in layer V was constituted by somatostatin-expressing regular spiking cells. When tested with 10 µM nicotine, these displayed larger somatic nicotinic currents in transgenic mice. Thus, during wakefulness, activation of ß2V287L-containing nAChRs by the high cholinergic tone may counteract hyperexcitability by promoting local inhibition by somatostatin-expressing cells and decreasing the effect of glutamatergic deficit in FS neurons. This interpretation was tested in networks disinhibited by 2 µM bicuculline. Slices expressing ß2V287L were more susceptible to develop synchronized activity in the absence of nicotine. Addition of the drug boosted excitability in the controls, but had little effect in ß2V287L. Our findings suggest why NREM sleep favors ADSHE seizures and nicotine can be palliative in patients.

Human airway organoids and microplastic fibers: A new exposure model for emerging contaminants.

Three-dimensional (3D) structured organoids are the most advanced in vitro models for studying human health effects, but their application to evaluate the biological effects associated with microplastic exposure was neglected until now. Fibers from synthetic clothes and fabrics are a major source of airborne microplastics, and their release from dryer machines is poorly understood. We quantified and characterized the microplastic fibers (MPFs) released in the exhaust filter of a household dryer and tested their effects on airway organoids (1, 10, and 50 µg mL-1) by optical microscopy, scanning electron microscopy (SEM), confocal microscopy and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). While the presence of MPFs did not inhibit organoid growth, we observed a significant reduction of SCGB1A1 gene expression related to club cell functionality and a polarized cell growth along the fibers. The MPFs did not cause relevant inflammation or oxidative stress but were coated with a cellular layer, resulting in the inclusion of fibers in the organoid. This effect could have long-term implications regarding lung epithelial cells undergoing repair. This exposure study using human airway organoids proved suitability of the model for studying the effects of airborne microplastic contamination on humans and could form the basis for further research regarding the toxicological assessment of emerging contaminants such as micro- or nanoplastics.

The Association between α-Synuclein and α-Tubulin in Brain Synapses.

α-synuclein is a small protein that is mainly expressed in the synaptic terminals of nervous tissue. Although its implication in neurodegeneration is well established, the physiological role of α-synuclein remains elusive. Given its involvement in the modulation of synaptic transmission and the emerging role of microtubules at the synapse, the current study aimed at investigating whether α-synuclein becomes involved with this cytoskeletal component at the presynapse. We first analyzed the expression of α-synuclein and its colocalization with α-tubulin in murine brain. Differences were found between cortical and striatal/midbrain areas, with substantia nigra pars compacta and corpus striatum showing the lowest levels of colocalization. Using a proximity ligation assay, we revealed the direct interaction of α-synuclein with α-tubulin in murine and in human brain. Finally, the previously unexplored interaction of the two proteins in vivo at the synapse was disclosed in murine striatal presynaptic boutons through multiple approaches, from confocal spinning disk to electron microscopy. Collectively, our data strongly suggest that the association with tubulin/microtubules might actually be an important physiological function for α-synuclein in the synapse, thus suggesting its potential role in a neuropathological context.

Phospho-HDAC6 Gathers Into Protein Aggregates in Parkinson's Disease and Atypical Parkinsonisms.

HDAC6 is a unique histone deacetylase that targets cytoplasmic non-histone proteins and has a specific ubiquitin-binding activity. Both of these activities are required for HDAC6-mediated formation of aggresomes, which contain misfolded proteins that will ultimately be degraded via autophagy. HDAC6 deacetylase activity is increased following phosphorylation on serine 22 (phospho-HDAC6). In human, HDAC6 localizes in neuronal Lewy bodies in Parkinson's disease (PD) and in oligodendrocytic Papp-Lantos bodies in multiple system atrophy (MSA). However, the expression of phospho-HDAC6 in post-mortem human brains is currently unexplored. Here, we evaluate and compare the distribution of HDAC6 and its phosphorylated form in human brains obtained from patients affected by three forms of parkinsonism: two synucleinopathies (PD and MSA) and a tauopathy (progressive supranuclear palsy, PSP). We find that both HDAC6 and its phosphorylated form localize with pathological protein aggregates, including α-synuclein-positive Lewy bodies in PD and Papp-Lantos bodies in MSA, and phospho-tau-positive neurofibrillary tangles in PSP. We further find a direct interaction of HDAC6 with α-synuclein with proximity ligation assay (PLA) in neuronal cell of PD patients. Taken together, our findings suggest that both HDAC6 and phospho-HDAC6 regulate the homeostasis of intra-neuronal proteins in parkinsonism.

Proliferative cells in the rat developing neocortical grey matter: new insights into gliogenesis.

The postnatal brain development is characterized by a substantial gain in weight and size, ascribed to increasing neuronal size and branching, and to massive addition of glial cells. This occurs concomitantly to the shrinkage of VZ and SVZ, considered to be the main germinal zones, thus suggesting the existence of other germinative niches. The aim of this study is to characterize the cortical grey matter proliferating cells during postnatal development, providing their stereological quantification and identifying the nature of their cell lineage. We performed double immunolabeling for the proliferation marker Ki67 and three proteins which identify either astrocytes (S100ß) or oligodendrocytes (Olig2 and NG2), in addition to a wider panel of markers apt to validate the former markers or to investigate other cell lineages. We found that proliferating cells increase in number during the first postnatal week until P10 and subsequently decreased until P21. Cell lineage characterization revealed that grey matter proliferating cells are prevalently oligodendrocytes and astrocytes along with endothelial and microglial cells, while no neurons have been detected. Our data showed that astrogliogenesis occurs prevalently during the first 10 days of postnatal development, whereas contrary to the expected peak of oligodendrogenesis at the second postnatal week, we found a permanent pool of proliferating oligodendrocytes enduring from birth until P21. These data support the relevance of glial proliferation within the grey matter and could be a point of departure for further investigations of this complex process.

The interactions of fullerene C60 and Benzo(α)pyrene influence their bioavailability and toxicity to zebrafish embryos.

This study aimed to assess the toxicological consequences related to the interaction of fullerene nanoparticles (C60) and Benzo(α)pyrene (B(α)P) on zebrafish embryos, which were exposed to C60 and B(α)P alone and to C60 doped with B(α)P. The uptake of pollutants into their tissues and intra-cellular localization were investigated by immunofluorescence and electron microscopy. A set of biomarkers of genotoxicity and oxidative stress, as well as functional proteomics analysis were applied to assess the toxic effects due to C60 interaction with B(α)P. The carrier role of C60 for B(α)P was observed, however adsorption on C60 did not affect the accumulation and localization of B(α)P in the embryos. Instead, C60 doped with B(α)P resulted more prone to sedimentation and less bioavailable for the embryos compared to C60 alone. As for toxicity, our results suggested that C60 alone elicited oxidative stress in embryos and a down-regulation of proteins involved in energetic metabolism. The C60 + B(α)P induced cellular response mechanisms similar to B(α)P alone, but generating greater cellular damages in the exposed embryos.

Characterization of PF4-Heparin Complexes by Photon Correlation Spectroscopy and Zeta Potential.

Heparin-induced thrombocytopenia (HIT) is associated with antibodies to complexes between heparin and platelet factor 4 (PF4), a basic protein usually found in platelet alpha granules. Heparin-induced thrombocytopenia antibodies preferentially recognize macromolecular complexes formed between positively charged PF4 and polyanionic heparins over a narrow range of molar ratios. The aim of this work was to study the complexes that human PF4 forms with heparins from various species, such as porcine, bovine, and ovine; heparins from various organs, such as mucosa and lung; and different low-molecular-weight heparins (LMWHs) at several stoichiometric ratios to evaluate their sizes and charges by photo correlation spectroscopy and zeta potential measurements. The resulting data of the PF4 complexes with unfractionated heparins (UFHs), LMWHs and their fractions, and oligosaccharide components suggest that the size of aggregates is not only a simple function of average molecular weight but also of the molecular weight distribution of the sample. Moreover, it was found that lower concentrations of the tested ovine-derived mucosal heparin are required to form the large PF4/heparin complexes as compared to mucosal porcine and bovine heparin.

Kainate receptor RNA editing is markedly altered by acute spinal cord injury.

We have previously observed changes in the RNA editing of AMPA receptors after acute spinal cord injury (SCI); this implies that post-transcriptional modifications are capable of affecting the physiological properties of glutamate receptor channels and related signal transduction in this neurodegenerative condition. Here, we report that the editing of the ionotropic KAR is markedly decreased at both GluK1 and GluK2 Q/R sites in the epicenter of the lesion and with distinct magnitude and kinetics also in the caudal and rostral portions of the injured cord. These effects are persistent, being observed as late as 30 days after lesioning. In addition, also the I/V and Y/C sites of GluK2 were severely affected after SCI. These findings add novel information to the relevance of editing of glutamate receptors following acute SCI, thus expanding the recently emerged role of post-transcriptional mechanisms under these experimental conditions.

The use of poly(N-[2-hydroxypropyl]-methacrylamide) hydrogel to repair a T10 spinal cord hemisection in rat: a behavioural, electrophysiological and anatomical examination.

There have been considerable interests in attempting to reverse the deficit because of an SCI (spinal cord injury) by restoring neural pathways through the lesion and by rebuilding the tissue network. In order to provide an appropriate micro-environment for regrowing axotomized neurons and proliferating and migrating cells, we have implanted a small block of pHPMA [poly N-(2-hydroxypropyl)-methacrylamide] hydrogel into the hemisected T10 rat spinal cord. Locomotor activity was evaluated once a week during 14 weeks with the BBB rating scale in an open field. At the 14th week after SCI, the reflexivity of the sub-lesional region was measured. We also monitored the ventilatory frequency during an electrically induced muscle fatigue known to elicit the muscle metaboreflex and increase the respiratory rate. Spinal cords were then collected, fixed and stained with anti-ED-1 and anti-NF-H antibodies and FluoroMyelin. We show in this study that hydrogel-implanted animals exhibit: (i) an improved locomotor BBB score, (ii) an improved breathing adjustment to electrically evoked isometric contractions and (iii) an H-reflex recovery close to control animals. Qualitative histological results put in evidence higher accumulation of ED-1 positive cells (macrophages/monocytes) at the lesion border, a large number of NF-H positive axons penetrating the applied matrix, and myelin preservation both rostrally and caudally to the lesion. Our data confirm that pHPMA hydrogel is a potent biomaterial that can be used for improving neuromuscular adaptive mechanisms and H-reflex responses after SCI.

Adult neural precursors isolated from post mortem brain yield mostly neurons: an erythropoietin-dependent process.

This study was aimed at the isolation of neural precursor cells (NPCs) capable of resisting to a prolonged ischemic insult as this may occur at the site of traumatic and ischemic CNS injuries. Adult mice were anesthetized and then killed by cervical dislocation. The cadavers were maintained at room temperature or at 4°C for different time periods. Post mortem neural precursors (PM-NPCs) were isolated, grown in vitro and their differentiation capability was investigated by evaluating the expression of different neuronal markers. PM-NPCs differentiate mostly in neurons, show activation of hypoxia-inducible factor-1 and MAPK, and express both erythropoietin (EPO) and its receptor (EPO-R). The exposure of PM-NPCs to neutralizing antibodies to EPO or EPO-R dramatically reduced the extent of neuronal differentiation to about 11% of total PM-NPCs. The functionality of mTOR and MAPK is also required for the expression of the neuronal phenotype by PM-NPCs. These results suggest that PM-NPCs can be isolated from animal cadaver even several hours after death and their self-renewable capability is comparable to normal neural precursors. Differently, their ability to achieve a neural phenotype is superior to that of NPCs, and this is mediated by the activation of hypoxia-induced factor 1 and EPO signaling. PM-NPCs may represent good candidates for transplantation studies in animal models of neurodegenerative diseases.

Acute spinal cord injury persistently reduces R/G RNA editing of AMPA receptors.

Spinal cord injury (SCI) triggers a complex ischemic and inflammatory reaction, involving activation of neurotransmitter systems, in particular glutamate, culminating in cell death. We hypothesized that SCI might lead to alteration in the RNA editing of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors that govern critical determinants of neuronal survival. To this end, we examined the molecular changes set in motion by SCI that affect the channel properties of AMPA receptors. SCI strongly reduced the level of AMPA receptor R/G editing, involving not only the site of the lesion but also adjacent areas of the cord spared by the lesion. The effects, with changes for some subunits and loci, were observed as long as 30 days after lesioning and may correlate with a partial decrease in enzymatic activity of adenosine deaminase acting on RNA 2 (ADAR2), as deduced from the analysis of ADAR2 self-editing. The reduced editing at the R/G site of glutamate receptor subunits (GluRs) is likely to reduce post-synaptic excitatory responses to glutamate, thus limiting the progression of cell death; however, prolonged suppression of GluR function in later stages may hinder synaptic plasticity. These observations provide the first direct evidence of the potential contribution of RNA editing to excitatory neural injury and recovery after SCI.

Embryonic stem cells promote motor recovery and affect inflammatory cell infiltration in spinal cord injured mice.

The purpose of this study was to determine the fate and the effects of undifferentiated embryonic stem cells (ESCs) in mice after contusive lesion of the spinal cord (SCI). Reproducible traumatic lesion to the cord was performed at T8 level by means of the Infinite Horizon Device, and was followed by intravenous injection of one million of undifferentiated ESCs through the tail vein within 2 h from the lesion. The ESCs-treated animals showed a significant improvement of the recovery of motor function 28 days after lesion, with an average score of 4.61+/-0.13 points of the Basso Mouse Scale (n=14), when compared to the average score of vehicle treated mice, 3.58+/-0.23 (n=10). The number of identified ESCs found at the lesion site was 0.6% of the injected cells at 1 week after transplantation, and further reduced to 0.04% at 1 month. It is, thus, apparent that the promoted hind-limb recovery cannot be correlated to a substitution of the lost tissue performed by the exogenous ESC. The extensive evaluation of production of several neuroprotective and inflammatory cytokines did not reveal any effect by ESC-treatment, but unexpectedly the number of invading macrophages and neutrophils was greatly reduced. This may explain the improved preservation of lesion site ventral myelin, at both 1 week (29+/-11%) and 1 month (106+/-14%) after injury. No teratoma formation was observed, although an inappropriate colonization of the sacral cord by differentiated nestin- and beta-tubulin III-positive ESCs was detected.

Viability-dependent promoting action of adult neural precursors in spinal cord injury.

The aim of the study was the assessment of the effects of adult neural stem cell (NSC) transplantation in a mouse model of spinal cord injury (SCI). The contusion injury was performed by means of the Infinite Horizon Device to allow the generation of reproducible traumatic lesion to the cord. We administered green fluorescent-labeled (GFP-)NSCs either by intravenous (i.v.) injection or by direct transplantation into the spinal cord (intraspinal route). We report that NSCs significantly improved recovery of hind limb function and greatly attenuated secondary degeneration. The i.v. route of NSC administration yielded better recovery than the intraspinal route of administration. About 2% of total i.v.-administered NSCs homed to the spinal cord injury site, and survived almost undifferentiated; thus the positive effect of NSC treatment cannot be ascribed to damaged tissue substitution. The NSCs homing to the injury site triggered, within 48 h, a large increase of the expression of neurotrophic factors and chemokines. One wk after transplantation, exogenous GFP-NSCs still retained their proliferation potential and produced neurospheres when recovered from the lesion site and cultured in vitro. At a later time, GFP-NSC were phagocytated by macrophages. We suggest that the process of triggering the recovery of function might be strongly related to the viability of GFP-NSC, still capable ex vivo of producing neurospheres, and their ability to modify the lesion environment in a positive fashion.

Single exposure to erythropoietin modulates Nerve Growth Factor expression in the spinal cord following traumatic injury: comparison with methylprednisolone.

Acute lesions of the spinal cord lead to dramatic changes in neuronal function. In the present study, we examined the possible involvement of neurotrophic factors in the action of the drug of choice for the treatment of such an emergency, i.e. the glucocorticoid methylprednisolone is compared to erythropoietin, a cytokine recently shown to markedly shorten the time necessary for motor recovery following injury [Gorio, A., Gokmen, N., Erbayraktar, S., Yilmaz, O., Madaschi, L., Cichetti, C., Di Giulio, A.M., Vardar, E., Cerami, A., Brines, M., 2002. Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma. Proc. Natl. Acad. Sci. 99, 9450-9455]. We found that methylprednisolone reduces the lesion-enhanced Nerve Growth Factor (NGF) mRNA levels 3 h after injury in the trauma epicenter and caudal section of the cord whereas erythropoietin reinforced the NGF gene expression. Three days after the occurrence of the lesion, erythropoietin, but not methylprednisolone, significantly up-regulated the NGF gene expression both caudally and rostrally to the lesion site, an effect that, based on the chemo-attractant properties of neurotrophin, might facilitate the growth of injured axons toward NGF-rich sites and contribute to the enhancement of the regenerative process. The differences between the effects of methylprednisolone and erythropoietin dissipate 7 days after the lesion when they both enhance NGF mRNA levels at the epicenter. These data show that methylprednisolone and erythropoietin display a different pattern of activation of the neurotrophin NGF which is strictly dependent on the portion of the cord examined and the time elapsed from the injury. Based on our results, we suggest that the higher increase of NGF expression mediated by erythropoietin soon after the injury might explain, at least in part, the improved recovery of motor functions produced by erythropoietin compared to methylprednisolone and saline.

Reparixin, an inhibitor of CXCR2 function, attenuates inflammatory responses and promotes recovery of function after traumatic lesion to the spinal cord.

It has been shown that the blockade of CXCR1 and CXCR2 receptors prevents ischemia/reperfusion damage in several types of vascular beds. Reparixin is a recently described inhibitor of human CXCR1/R2 and rat CXCR2 receptor activation. We applied reparixin in rats following traumatic spinal cord injury and determined therapeutic temporal and dosages windows. Treatment with reparixin significantly counteracts secondary degeneration by reducing oligodendrocyte apoptosis, migration to the injury site of neutrophils and ED-1-positive cells. The observed preservation of the white matter might also be secondary to the enhanced proliferation of NG2-positive cells. The expression of macrophage-inflammatory protein-2, tumor necrosis factor-alpha, interleukin (IL)-6, and IL-1 beta was also counteracted, and the proliferation of glial fibrillary acidic protein-positive cells was markedly reduced. These effects resulted in a smaller post-traumatic cavity and in a significantly improved recovery of hind limb function. The best beneficial outcome of reparixin treatment required 7-day administration either by i.p. route (15 mg/kg) or subcutaneous infusion via osmotic pumps (10 mg/kg), reaching a steady blood level of 8 microg/ml. Methylprednisolone was used as a reference drug; such treatment reduced cytokine production but failed to affect the rate of hind limb recovery.

Methylprednisolone neutralizes the beneficial effects of erythropoietin in experimental spinal cord injury.

Inflammation plays a major pathological role in spinal cord injury (SCI). Although antiinflammatory treatment using the glucocorticoid methyprednisolone sodium succinate (MPSS) improved outcomes in several multicenter clinical trials, additional clinical experience suggests that MPSS is only modestly beneficial in SCI and poses a risk for serious complications. Recent work has shown that erythropoietin (EPO) moderates CNS tissue injury, in part by reducing inflammation, limiting neuronal apoptosis, and restoring vascular autoregulation. We determined whether EPO and MPSS act synergistically in SCI. Using a rat model of contusive SCI, we compared the effects of EPO [500-5,000 units/kg of body weight (kg-bw)] with MPSS (30 mg/kg-bw) for proinflammatory cytokine production, histological damage, and motor function at 1 month after a compression injury. Although high-dose EPO and MPSS suppressed proinflammatory cytokines within the injured spinal cord, only EPO was associated with reduced microglial infiltration, attenuated scar formation, and sustained neurological improvement. Unexpectedly, coadministration of MPSS antagonized the protective effects of EPO, even though the EPO receptor was up-regulated normally after injury. These data illustrate that the suppression of proinflammatory cytokines alone does not necessarily prevent secondary injury and suggest that glucocorticoids should not be coadministered in clinical trials evaluating the use of EPO for treatment of SCI.

Muscle reinnervation and IGF-I synthesis are affected by exposure to heparin: an effect partially antagonized by anti-growth hormone-releasing hormone.

Sciatic nerve crush was performed in 2-day-old rats, then reinnervation of the extensor digitorum longus muscle, motor neuron survival, and muscle IGF-I production were monitored. In saline-treated rats, the extent of reinnervation was around 50% and the number of EDL reinnervating motor neurons was significantly reduced. In heparin-treated rats the extent of muscle reinnervation, the recovery of nerve-evoked muscle twitch tension, and the number of motor neurons reinnervating the extensor digitorum longus muscle were greatly enhanced compared to saline-treated rats. In addition, treatment with heparin increased markedly insulin-like growth factor-I levels in denervated muscles. The concomitant exposure to anti-growth hormone releasing hormone partially abolished the stimulatory action of heparin on muscle reinnervation and prevented the increase of insulin-like growth factor-I muscle levels.

Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma.

Erythropoietin (EPO) functions as a tissue-protective cytokine in addition to its crucial hormonal role in red cell production. In the brain, for example, EPO and its receptor are locally produced, are modulated by metabolic stressors, and provide neuroprotective and antiinflammatory functions. We have previously shown that recombinant human EPO (rhEPO) administered within the systemic circulation enters the brain and is neuroprotective. At present, it is unknown whether rhEPO can also improve recovery after traumatic injury of the spinal cord. To evaluate whether rhEPO improves functional outcome if administered after cord injury, two rodent models were evaluated. First, a moderate compression of 0.6 N was produced by application of an aneurysm clip at level T3 for 1 min. RhEPO (1,000 units per kg of body weight i.p.) administered immediately after release of compression was associated with partial recovery of motor function within 12 h after injury, which was nearly complete by 28 days. In contrast, saline-treated animals exhibited only poor recovery. In the second model used, rhEPO administration (5,000 units per kg of body weight i.p. given once 1 h after injury) also produced a superior recovery of function compared with saline-treated controls after a contusion of 1 N at level T9. In this model of more severe spinal cord injury, secondary inflammation was also markedly attenuated by rhEPO administration and associated with reduced cavitation within the cord. These observations suggest that rhEPO provides early recovery of function, especially after spinal cord compression, as well as longer-latency neuroprotective, antiinflammatory and antiapoptotic functions.

Co-administration of IGF-I and glycosaminoglycans greatly delays motor neurone disease and affects IGF-I expression in the wobbler mouse: a long-term study.

The study on wobbler mouse has shown that the combined treatment with low doses of glycosaminoglycans (GAGs) and insulin-like growth factor-I (IGF-I) fully prevented motor neurone death and forelimb impairment up to 9-12 weeks of a mouse's life. The effect was accompanied by the prevention of the early hypertrophy of wobbler neurones, an effect likely due to the promotion of neuronal survival. At the 18th week, wobbler mice treated with IGF-I + GAGs still showed significantly improved forelimb function, reduced muscle atrophy and a higher number of cervical motor neurones. IGF-I alone and GAGs alone were active up to the 3rd week of treatment; thereafter the beneficial effects of single treatments decreased drastically. GAGs and IGF-I treatments also affected IGF-I plasma and muscle levels. In wobbler mice there was a progressive reduction in IGF-I plasma levels that was prevented by IGF-I or GAGs alone and greatly increased, even above heterozygote levels, by the combination treatment. Such a powerful increase was correlated by a small enhancement in insulin-like growth factor binding protein-3 (IGFBP-3) plasma levels, while treatment with IGF-I alone affected very significantly both IGFBP-1 and IGFBP-3. Co-treatment also prevented the decrease in IGF-I content observed in vehicle-treated wobbler mice forelimb muscles.