key: cord-0741839-ywa7wzs7
authors: Giardino, L.; Giuliani, A.; Fernandez, M.; Calzà, L.
title: Spinal motoneurone distress during experimental allergic encephalomyelitis
date: 2004-05-04
journal: Neuropathol Appl Neurobiol
DOI: 10.1111/j.1365-2990.2004.00559.x
sha: d2251548f5eb2e0bd950ac2df1de4a5abbdd5440
doc_id: 741839
cord_uid: ywa7wzs7

The main pathophysiological feature characterizing multiple sclerosis (MS) is demyelination. However, the possibility of neural damage has recently been proposed as a mechanism in chronic disease. Experimental allergic encephalomyelitis (EAE) is the most widely used experimental model for MS. We investigated occurrences of microglial activation and astrocytosis in the spinal cord, choline acetyl‐transferase (ChAT) and calcitonin gene‐related peptide (CGRP) mRNA regulation in spinal motoneurones during EAE. EAE was induced in female Lewis rats by injecting guinea pig spinal cord tissue in complete Freund's adjuvant (CFA) to which heat‐inactivated Mycobacterium had been added. Rats injected with CFA and uninjected rats were used as controls. ChAT and CGRP mRNAs were studied by in situ hybridization in the lumbar spinal cord and a computerized grain counting procedure was used for quantification. No differences in ChAT mRNA level were found between control and CFA‐injected rats. ChAT mRNA level was strongly reduced in EAE 14 days after immunization and then recovered (29 days after immunization). CGRP mRNA increased 14 days after immunization, and then recovered to control level. Extensive long‐lasting gliosis developed in the spinal cord and around motoneurones and a transient expression of p75(LNGFR) in motoneurones was also found. These data suggest that during EAE, gliosis induces distress in spinal cord neurones involving the synthesis enzyme for the main transmitter.

Multiple sclerosis (MS) is a demyelinating disease characterized by extensive inflammation and gliosis in the central nervous system [1] . The main pathological feature of MS is widespread demyelination and oligodendrocyte degeneration [2] . MS has therefore been interpreted as an oligodendrocyte disease. More recently, the possibility of neural damage has been raised. In particular, it is accepted that axonal loss occurs in different areas of the central nervous system [3, 4] including the spinal cord [5] and corpus callosum [6] ; this lesion is involved in permanent disability characterizing the later chronic progressive stage of MS [7] [8] [9] . Magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS) and MRS imaging studies have provided evidence that axonal loss and neural damage in MS can be both substantial and early [10, 13] and these noninvasive measures have demonstrated the existence of direct links between these axonal changes and disability [14] [15] [16] .

Histopathological studies have confirmed this clinical evidence, indicating that both atrophy and decreased density contribute to substantial axonal loss in brain and spinal cord [5, 17] and that the highest incidence of acute axonal injury is found during active demyelination [18] , as well as in patients with secondary progressive MS [3] .

Evidence from work on animals has also demonstrated that damage to axons can occur with central nervous system inflammation, as indicated in mice infected with a neurotropic coronavirus [19] , in experimental allergic encephalomyelitis (EAE) [18, 20, 21] and in nonhuman primate models of MS [22] .

Neuroprotection has consequently come to be considered a possible strategy for MS patients [8, 23] . However, development of neuroprotective strategies is dependent on defining the precise mechanism whereby effector cells and molecules damage axons, and on whether neural damage is limited to the axon or also involves the soma. On the basis of behavioural data indicating severe neurological disabilities, including force deficit and diffuse tissue inflammation and gliosis in the spinal cord of rats affected by EAE, we investigated the relation between clinical profile, gliosis and motoneurone neurochemical phenotype in the spinal cord of EAE-affected rats. We focused on the acetylcholine synthesis enzyme [choline acetyltransferase (ChAT)], calcitonin gene-related peptide (CGRP), which is considered a trophic peptide for motoneurones, and nerve growth factor (NGF) low-affinity receptor p75 LNGFR , which normally is not expressed by motoneurones in adult animals. Alterations of all these markers have been described in different experimental conditions associated with axonal damage [24] .

Female pathogen-free Lewis rats, 150-175 g body weight (Charles River, Italy) were used. A group of rats was sensitized with a medium containing 0.15 g/ml guinea pig spinal cord tissue in complete Freund's adjuvant (CFA, Sigma, St. Louis, MO, USA), 50% v/v to which 5 mg/ml of heat-inactivated Mycobacterium (H37Ra Difco, Detroit, MI, USA) was added. A further group of rats was injected with CFA + heat-inactivated Mycobacterium. Uninjected rats were also used as controls. Rats were regularly weighed and examined for clinical signs of EAE up to 79 days after immunization by a trained observer and scored on a neurological disability scale according to which grade 1 = loss of tail tone; grade 2 = weakness in one or both hind legs or middle ataxia; grade 3 = ataxia or paralysis; grade 4 = severe hind leg paralysis; grade 5 = severe hind leg paralysis accompanied by urinary incontinence. Uninjected and CFA-injected rats were rated as O in all observations. In all experiments, groups of EAE animals were sacrificed at 14, 21 and 79 days after immunization, adjuvant-injected animals at 14 and 79 days after sensitization, and control animals 79 days after inclusion in the experiment.

All animal protocols described herein were carried out according to the European Community Council Directives of 24 November 1986 (86/609/EEC) and approved by our intramural committee and the Italian Health Ministry, in compliance with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals .

For immunocytochemical experiments, five to six animals in each experimental group were sacrificed at set times. Animals deeply anaesthetized with ketamine (Ketalar, Parke Davis, Italy) 10 mg/kg of body weight, i.p. + diazepam 2 mg/kg, i.m., were perfused through the ascending aorta with 100 ml (50 ml at 37 ∞ C, 50 ml ice cold) Tyrode-Ca ++ , pH 6.9, followed by 4% paraformaldehyde in Sorensen phosphate buffer 0.1 M pH 7.0 for 25-30 min. During perfusion the animals were bathed in ice-cold water. The brain and spinal cord were then removed and immersed for 2 h in ice-cold fixative, before rinsing for 48 h in ice-cold 0.1 M phosphate buffer. For immunocytochemistry, the following antisera were used: mouse antiglial fibrillary acid protein (GFAP) (Chemicon Temecula, CA, USA), mouse anti-nestin (BD PharMingen, San Jose, CA, USA), mouse anti-rat CD11b (OX42, Sera-Laboratory, Crawley Down, UK), mouse anti-oligodendrocytes (Chemicon Temecula; immunogen: oligodendrocytes cultured by the rat olfactory bulb), goat antip75 LNGFR (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). For immunofluorescence experiments, tissue was fast frozen in CO 2 and cut on a Leitz cryostat (thickness of section 14 m m, -20 ∞ C). Sections were first incubated in 0.1 M phosphate-buffered saline (PBS) at room temperature for 10-30 min, followed by incubation at 4 ∞ C for 48 h in a humid atmosphere with the primary antibody diluted in PBS containing 0.3% Triton X-100, v/v. Staining specificity was assessed by in vitro overnight preincubation of the antiserum with the respective antigen (100 m g of antigen/ ml diluted antiserum). This treatment prevented staining. After rinsing in PBS for 30 min (3 ¥ 10 min), the sections were incubated at 37 ∞ C for 120 min in a humid atmosphere with fluoresceine isothiocyanate-conjugated antimouse or anti-goat immunoglobulin (Dako, Glostrup, Denmark) containing 0.3% Triton X-100. For double experiments, two primary antisera raised in different animal species and appropriate secondary antisera were applied concomitantly. Sections were then rinsed in PBS (as above), mounted in glycerol and PBS (3 : 1, v/v) containing 1,4-phenylendiamine, 0.1 g/l. They were then examined using a Nikon Microphot FXA microscope.

For the avidin-biotin complex (ABC) technique, vibratome sections (50 m m thickness) were immediately processed for the ABC technique according to a freefloating procedure. In each experiment, sections from all animals were run in the same assay. Sections were first incubated in 0.1 M PBS at room temperature for 10-30 min, pretreated with H 2 O 2 to quench endogenous peroxidase activity and then incubated with 1.5-2.0% normal serum, followed by overnight incubation at 4 ∞ C with the primary antisera diluted in PBS containing 0.3% Triton X-100, v/v. Sections were rinsed in PBS for 30 min (3 ¥ 10 min) and then incubated with biotinylated antimouse or antigoat immunoglobulin (Dako); they were rinsed again in PBS and finally incubated using streptavidin biotinylated horseradish peroxidase complex (Amersham, Little Chalfont, UK) 1 : 250. Diaminobenzidine (0.5 mg/ml in Tris HCl 0.1 M pH 7.5 + H 2 O 2 0.03%) was used to detect the immunocomplex. Preparations were then examined using a Nikon Microphot FXA microscope and Tmax 400 film (Kodak, Hemel Hempstead, UK) was used for photography. Color slides were then loaded via Nikon SuperCool Scan 4000 and Figure 2 was generated using Adobe Photoshop 6.0 software.

Quantification of immunocytochemical staining for glial reaction was carried out on an immunofluorescence preparation using the analytical imaging station (AIS) (Imaging Research Inc., St. Catharines, Canada). Three animal/group/times were used and six sections from the lumbar spinal cord were analysed in each animal. In each case, the ventral horn on both sides, including layers 8 and 9, was analysed in order to evaluate the percentage immunoreactive area of each antigen [25, 26] . Briefly, a contrast-enhancing procedure was first applied to clearly identify the edge of immunoreactive cells. A threshold procedure was then applied to sample the immunoreactive area over the entire sampled area (% area). The average values from each animal were used for statistical analysis. Spinal cord inflammation was then scored on the same section after toluidine blue contro-staining according to Hickey et al . [27] using a previously described computerized image analysis procedure [28] in which 1 = occasional mononuclear cells; 2 = sparse cellular infiltrate; 3 = large patches of infiltrate with numerous mononuclear cells; 4 = dense accumulation of mononuclear cells.

Four to five animal/group/times were used for in situ hybridization experiments. Animals were sacrificed by decapitation, the spinal cord was quickly dissected out and frozen using CO 2 . Coronal sections were cut in a cryostat from L5 and collected on ProbeOn microscope slides (Fisher Scientific, Pittsburg, PA, USA). Oligonucleotide probes complementary to mRNAs encoding ChAT (nucleotides 1818-1860) and a CGRP 664-698, respectively, were synthesized in a Beckmann OLIGO 1000 DNA synthesizer (Fullerton, CA, USA). The oligonucleotide probes were labelled at the 3 ¢ -end with a -35 S-dATP (New England Nuclear, Boston, MA, USA) using terminal deoxynucleotidyltransferase (Amersham) in a buffer containing 10 mM CoCl 2 , 1 mM dithiothreitol (DTT), 300 mM Tris base, and 1.4 M potassium cacodylate (pH 7.2). Afterwards, the labelled probes were purified through Nensorb-20 columns (New England Nuclear), and DTT was added to obtain a final concentration of 10 mM. The resulting specific activity ranged from 1 to 4 ¥ 10 6 dpm/ng oligonucleotide. The sections were brought to room temperature, air dried, covered with a hybridization buffer containing 50% formamide, 4 ¥ SSC (1 ¥ SSC: 0.15 M NaCl, 0.015 M sodium citrate), 1 ¥ Denhardt's solution (0.02% polyvinyl-pyrrolidone, 0.02% bovine serum albumin and 0.02% Ficoll), 1% sarcosyl, 0.02 M phosphate buffer (pH 7.0), 10% dextran sulphate, 500 mg/ml heat-denatured salmon sperm DNA, and 200 mM DTT and 40 ng/ml of the labelled probes [29] . The slides were placed in a humid chamber and incubated for 15-20 h at 50 ∞ C. The sections were then rinsed in 1 ¥ SSC at 55 ∞ C for 1 h with six changes and washed in the same buffer for 1 h at room temperature. Finally, the slides were rinsed in distilled water followed by 60% and 95% ethanol (2 min each), air dried and then dipped in NTB2 nuclear track emulsion (Kodak) for 3 weeks before being developed with Kodak D19 for 3 min and fixed with G333 (Agfa Gevaert, Leverkusen, Germany) for 10 min. Quantitative analysis was performed on emulsion-dipped sections, slightly counterstained with toluidine blue using AIS (Imaging Research Inc.) grain counting software. ChAT and CGRP mRNAs expression was measured in the motoneurones on both sides in the ventral horn of the spinal cord, in an area corresponding to the lateral motor nucleus, by choosing five matching sections in each animal. Eight to 10 cells on each side were then measured. Cells were considered labelled if the silver grains were more than five times the background silver level, as determined by averaging grain counts over defined areas of the spinal cord devoid of positively labelled cell bodies. The average values from each animal were used for statistical analysis.

One-way analysis of variance ( ANOVA ) was used, followed by Dunnet's test and Student's t -test.

Clinical profiles expressing neurological disabilities in EAE and body weight of experimental animals are reported in Figure 1 . The severity of EAE gradually increases, reaches its peak between 8 and 14 days after immunization and then partially recovers. Body weight growth stops at a point corresponding to the acute phase of the disease. The growth curve then recovers but the body weight of the experimental animals remains significantly lower than control animals. In our experience [28] , as confirmed in this experiment, the disease relapses with lower severity in 60% of animals. Only relapsing animals have been included in the study.

As described in previous reports [28, 30] and confirmed in this study (images not shown), in only a few rats were small scattered areas of inflammation (1-2 according to Hickey et al .) [27] observed 14 days after immunization. Conversely, severe inflammatory cellular infiltrate, mainly composed by mononuclear cells, appeared in many areas of the brain and spinal cord between 14 and 20 days after myelin immunization. Almost all EAE animals showed a histological picture scored as 4. Histopathological examination of EAE relapsing animals evidenced multiple, confluent foci of inflammation in both the white and the grey matter of the spinal cord, where also necrosis was observed (scored 3-4 according to Hickey et al. 1983 ) [27] .

Glial cells in brain and spinal cord of EAE and control animals were studied using OX42 as marker for activated microglial cells, GFAP as marker for astrocytes and nestin as marker for activated and/or newly generated astrocytes ( Figure 2 ). Starting from day 14 after immunization, a disaggregation of white matter bundles in the spinal cord was observed in EAE animals, which worsened during clinical remission and relapse. Images in Figure 2 (Figure 2 G , H , N ) , appear as early as 14 days after immunization, for example, at a disease stage in which cellular inflammation is not yet pre-eminent (Figure 2 C ) , and then decline (Figure 2 D , 21 days after injection). GFAP-IR is higher in EAE animals 21 days after immunization than in controls and further increases in relapsing animals, where an intense astrocytosis is observed around motoneurones in the ventral horn (Figure 2 E , control; Figure 2 F , EAE 79 days after injection). Quantification of nestin-, OX42-and GFAPimmunostaining was performed in the ventral horn of the lumbar tract of the spinal cord. The quantitative results referring to the qualitative data described above are shown in Figure 3 . With the exception of a small but significant microglial activation in adjuvant-injected animals, no differences were observed between uninjected and adjuvant-injected rats.

Large neurones in the ventral horn of the spinal cord express ChAT and CGRP mRNAs. Quantitative analysis of ChAT and CGRP mRNAs levels in single motoneurones in control animals and during EAE is shown in Figure 4 .

ChAT mRNA level declines in the acute stage of EAE and remains lower than in control animals in the remission phase. No differences between pathological and control animals were observed at the end of the experiment, which corresponds to a partial recovery from relapse. Conversely, CGRP mRNA level is higher in motoneurones in pathological animals in the acute phase of the disease. Finally, p75 LNGFR -IR, which is never observed in control animals, appears transiently in motoneurones of pathological animals 14 days after immunization ( Figure 2 I -M ).

In this study we showed that during the inflammatoryautoimmune disease EAE spinal motoneurones undergo phenotype changes involving ChAT, CGRP and p75 simi-lar to those described following axonal lesion, which supports the view that long-lasting neurological disabilities are related to neurone alterations that follow demyelination. We also confirmed that a severe gliosis results from astrocyte hypertrophy and hyperplasy in EAE [1] . GFAP-IR cells surrounds the cell body and dendrites of spinal motoneurones during acute EAE, and we found that GFAP-immunostaining further increases after resolution of the acute phase. While axonal damage has been indicated as one of the typical features of MS and EAE histopathology [3, 15] ever since it was first described, the possibility of whole neurone damage being a possible substrate for both permanent motor disabilities and cognitive problems and degeneration has only emerged in recent years. Gene expression switching has been described in spinal motoneurones in response to axotomy and during regeneration [31, 32] . Sciatic nerve axotomy produces a transient loss of ChAT mRNA, re-expression of p75 LNGFR [24] and increase in cellular content of CGRP and mRNA [33] . Demyelinating lesions located where motor roots emerge from the spinal cord cause axonal lesions in motor neurones [18] . We describe how a decrease in ChAT mRNA expression is linked to increased CGRP mRNA levels and the re-appearance of p75 LNGFR -IR in the acute stage of EAE. ChAT mRNA levels decrease concomitantly with dramatic microglial (OX42-IR) and astroglial activation, as described by nestin-IR. Nestin is recognized as an earlier marker for astrocytic activation than GFAP [34] . CGRP in motoneurone acts as cotransmitter for acetylcholine, enhancing its spontaneous release in skeletal muscle, but also has a perhaps prevalent trophic action [35] . The rise of CGRP-IR in deafferentiated motoneurones is generally attributed to increased synthesis, although accumulation because of the blocking of peripheral transport cannot be excluded [36, 37] . The neurotrophin receptor p75 LNGFR , which binds NGF, brain-derived neurotrophic factor (BDNF), neurotrophin 3-4 (NT3-4) and tumour necrosis factor-a (TNF-a ) [38] , is expressed by developing motoneurones in the spinal cord, whereas in adult motoneurones it re-appears after lesions, including sciatic nerve axotomy [39, 40] . p75 LNGFR has also been associated with cell death [41] . However, it is accepted that p75 LNGFR upregulation possibly increases resistance to pathological motor neurone degeneration [42] and is present only in adult motor neurones that are allowed to regenerate [43] .

Acute axonal damage is associated with an axonal conduction block, ion channel redistribution [20] and interruption of axoplasmic flow [44] , including also trophic factors. We have already reported a dramatic drop in NGF content in the spinal cord during the acute phase of EAE and this defect could be involved in the clinical symptoms [30] . Indeed NGF administration attenuates neurological deficits in EAE marmosets [45, 46] , and ciliary neurotrophic factor (CNTF), which has been indicated as a major trophic factor for adult motoneurones, is a protective factor for severe neurological deficits in EAE [47] . Finally, a proper axonal function is required for successful remyelination in experimental pathologies [48] . Electrical activity in the axon is actually involved in regulating differentiation and the survival of oligodendrocyte precursors during development [49] and plays a key role in the induction of myelination [50] .

Changes in neurochemical phenotype in spinal motoneurones could be related to the axonal damage at the point of emergence of the motor root in the extensive demyelinating regions present in the spinal cord during EAE, but also to other types of attack, for example, glutamate excitotoxicity, which is also related to permanent gliosis [51] . We found that immunostaining of an astrocyte marker protein such as GFAP clearly highlights the cell body and dendrites of motoneurones in layers 8 and 9 of the ventral horn of the spinal cord. Astrocytes in EAE animals actually have a decreased capacity to metabolize glutamate, as indicated by the severe decrease in the activity of specific enzymes [51] . Damage to dendrites and synapses occurs in motoneurones in inflamed spinal cord during EAE, as indicated by quantitative analysis of protein associated with these cell components [32] . Dendrite damage recovers significantly, although incompletely, during EAE remission [52] , such as molecular indices of synaptic structures [53] . The temporal profile of alterations in dendrite and synapse damage in spinal cord correlates with the neurochemical changes we describe in the present study. In spite of the recovery, we cannot exclude that repeated inflammatory attacks or permanent gliosis may lead to permanent phenotype changes and even to motoneurone degeneration. Because of either axonal pathology resulting from demyelination and consequent neuronal stress, or a direct inflammatory attack on the cell body, modifications of the neurochemical phenotype in single spinal motoneurones observed in acute EAE indicate that severe motoneurone distress occurs in early phases of the disease. Our study was not designed to evaluate motoneurone cell number, so we cannot speculate as to whether recovery of normal neurochemical phenotype in single neurones means recovery in all neurones or only in surviving neurones. Recently, Smith et al . [54] described how motoneurones are subject to lymphocyte attack leading to a 30% reduction in neurone density in the ventral spinal cord in acute EAE. Neural cell loss has been indicated as one of the possible causes of brain and spinal cord atrophy in MS and EAE [55] . Apoptosis actually causes severe cell loss secondary to axonal damage in retinal ganglion cells during EAE [56] and it has been shown that cerebrospinal fluid from MS patients induces neuronal apoptosis in vitro [57] . Imaging studies have indicated that while normal brain atrophy is estimated at around 0.1-0.2% per year in normal subjects, it increases to approximately 1% in patients with progressive MS [58, 59] . Brain and spinal cord atrophy is also a common feature in remittance-relapsing MS including also the spinal cord [60] .

In conclusion, our data further suggest that distress, or lesion or degeneration of selective neural populations including cholinergic motoneurones in the spinal cord correlates with a temporal profile of neurological disability of EAE and MS. Thus, not only axons, but also the whole neurone should be investigated in order to define a possible neuroprotective strategy to include in MS multitherapy [61] . 

The pathogenesis of demyelinating disease

Mechanisms of damage to myelin and oligodendrocytes and their relevance to disease

Acute axonal injury in multiple sclerosis. Correlation with demyelination and inflammation

Axonal damage in acute multiple sclerosis lesions

Spinal cord axonal loss in multiple sclerosis: a post-mortem study

Size-selective neuronal changes in the anterior optic pathways suggest a differential susceptibility to injury in multiple sclerosis

Axonal pathology in myelin disorders

Multiple sclerosis: more than inflammation and demyelination

Axonal degeneration in the pathogenesis of multiple sclerosis

Magnetic resonance spectroscopy: imaging axonal damage in MS

In vivo evidence for axonal dysfunction remote from focal cerebral demyelination of the type seen in multiple sclerosis

Evidence of axonal damage in the early stages of multiple sclerosis and its relevance to disability

Proton MR spectroscopy to assess axonal damage in multiple sclerosis and other white matter disorders

Investigation of MS normal-appearing brain using diffusion tensor MRI with clinical correlations

Axonal damage correlates with disability in patients with relapsing-remitting multiple sclerosis. Results of a longitudinal magnetic resonance spectroscopy study

Putting magnetic resonance spectroscopy studies in context: axonal damage and disability in multiple sclerosis

Matthews PM. Regional axonal loss in the corpus callosum correlates with cerebral white matter lesion volume and distribution in multiple sclerosis

Distribution of a calcium channel subunit in dystrophic axons in multiple sclerosis and experimental autoimmune encephalomyelitis

Axonal damage is T cell mediated and occurs concomitantly with demyelination in mice infected with a neurotropic coronavirus

Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions

Axonal degeneration is an early pathological feature in autoimmune-mediated demyelination in mice

Demyelination and axonal damage in a non-human primate model of multiple sclerosis

Mechanisms of tissue injury in multiple sclerosis: opportunities for neuroprotective therapy

Peripheral nerve regeneration and neurotrophic factors

Nerve growth factor control of neuronal expression of angiogenetic and vasoactive factors

Neuroprotection and aging of the cholinergic system: a role for the ergoline derivative nicergoline (Sermion)

Identification and quantitation of T lymphocyte subsets found in the spinal cord of the Lewis rat during acute experimental allergic encephalomyelitis

Further evidence for a role of nitric oxide in experimental allergic encephalomyelitis: aminoguanidine treatment modifies its clinical evolution

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

Timecourse changes of nerve growth factor, corticotropinreleasing hormone, and nitric oxide synthase isoforms and their possible role in the development of inflammatory response in experimental allergic encephalomyelitis

Potential roles of gene expression change in adult rat spinal motoneurons following axonal injury: a comparison among c-jun, off-affinity nerve growth factor receptor (LNGFR), and nitric oxide synthase (NOS)

Dendritic and synaptic pathology in experimental autoimmune encephalomyelitis

Fibroblast growth factors regulate calcitonin generelated peptide mRNA expression in rat motoneurons after lesion and in culture

Transient coexpression of nestin, GFAP, and vascular endothelial growth factor in mature reactive astroglia following neural grafting or brain wounds

The peptidergic motoneurone

Peripheral nerve section induces increased levels of calcitonin gene-related peptide (CGRP)-like immunoreactivity in axotomized motoneurons

Calcitonin gene-related peptide in rat spinal cord motoneurons: subcellular distribution and changes induced by axotomy

Neurotrophin signal transduction in the nervous system

Nerve growth factor receptor immunoreactivity in neurons of the normal adult rat spinal cord and its modulation after peripheral nerve lesions

Modulation of lowaffinity nerve growth factor receptor in injured adult rat spinal cord motoneurons

Induction of postnatal Schwann cell death by the low-affinity neurotrophin receptor in vitro and after axotomy

Experimental rationale for the therapeutic use of neurotrophins in amyotrophic lateral sclerosis

Re-expression of p75NTR by adult motor neurons after axotomy is triggered by retrograde transport of a positive signal from axons regrowing through damaged or denervated peripheral nerve tissue

Traumatically induced axonal injury: pathogenesis and pathological implications

Surprising pleiotropy of nerve growth factor in the treatment of experimental autoimmune encephalomyelitis

Human nerve growth factor protects common marmosets against autoimmune encephalomyelitis by switching the balance of T helper cell type 1 and 2 cytokines within the central nervous system

CNTF is a major protective factor in demyelinating CNS disease: a neurotrophic cytokine as modulator in neuroinflammation

The oligodendrocyte precursor cell in health and disease

Control of oligodendrocyte number in the developing rat optic nerve

Induction of myelination in the central nervous system by electrical activity

Glutamate metabolism is down-regulated in astrocytes during experimental allergic encephalomyelitis

Neuroprotection by encephalomyelitis. Rescue of mechanically injured neurons and neurotrophin production by CNSinfiltrating T and natural killer cells

Serotonergic neuronal sprouting as a potential mechanism of recovery in multiple sclerosis

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Acute neuronal apoptosis in a rat model of multiple sclerosis

Neuronal apoptosis induced by cerebrospinal fluid from multiple sclerosis patients correlates with hypointense lesions on T1 magnetic resonance imaging

Axonal and neuronal degeneration in multiple sclerosis: mechanisms and functional consequences

Brain atrophy in clinically early relapsingremitting multiple sclerosis

Brain and spinal cord atrophy in multiple sclerosis: role as a surrogate measure of disease progression

Multiple approaches to multiple sclerosis

This study was supported by Fondazione Cassa di Risparmio in Bologna and Pathophysiology Center for the Nervous System, Modena, Italy.