Mitochondrial damage and "plugging" of transport selectively in myelinated, small-diameter axons are major early events in peripheral neuroinflammation.

BACKGROUND: Small-diameter, myelinated axons are selectively susceptible to dysfunction in several inflammatory PNS and CNS diseases, resulting in pain and degeneration, but the mechanism is not known. METHODS: We used in vivo confocal microscopy to compare the effects of inflammation in experimental autoimmune neuritis (EAN), a model of Guillain-Barré syndrome (GBS), on mitochondrial function and transport in large- and small-diameter axons. We have compared mitochondrial function and transport in vivo in (i) healthy axons, (ii) axons affected by experimental autoimmune neuritis, and (iii) axons in which mitochondria were focally damaged by laser induced photo-toxicity. RESULTS: Mitochondria affected by inflammation or laser damage became depolarized, fragmented, and immobile. Importantly, the loss of functional mitochondria was accompanied by an increase in the number of mitochondria transported towards, and into, the damaged area, perhaps compensating for loss of ATP and allowing buffering of the likely excessive Ca2+ concentration. In large-diameter axons, healthy mitochondria were found to move into the damaged area bypassing the dysfunctional mitochondria, re-populating the damaged segment of the axon. However, in small-diameter axons, the depolarized mitochondria appeared to "plug" the axon, obstructing, sometimes completely, the incoming (mainly anterograde) transport of mitochondria. Over time (~ 2 h), the transported, functional mitochondria accumulated at the obstruction, and the distal part of the small-diameter axons became depleted of functional mitochondria. CONCLUSIONS: The data show that neuroinflammation, in common with photo-toxic damage, induces depolarization and fragmentation of axonal mitochondria, which remain immobile at the site of damage. The damaged, immobile mitochondria can "plug" myelinated, small-diameter axons so that successful mitochondrial transport is prevented, depleting the distal axon of functioning mitochondria. Our observations may explain the selective vulnerability of small-diameter axons to dysfunction and degeneration in a number of neurodegenerative and neuroinflammatory disorders.

Impulse conduction increases mitochondrial transport in adult mammalian peripheral nerves in vivo.

Matching energy supply and demand is critical in the bioenergetic homeostasis of all cells. This is a special problem in neurons where high levels of energy expenditure may occur at sites remote from the cell body, given the remarkable length of axons and enormous variability of impulse activity over time. Positioning mitochondria at areas with high energy requirements is an essential solution to this problem, but it is not known how this is related to impulse conduction in vivo. Therefore, to study mitochondrial trafficking along resting and electrically active adult axons in vivo, confocal imaging of saphenous nerves in anaesthetised mice was combined with electrical and pharmacological stimulation of myelinated and unmyelinated axons, respectively. We show that low frequency activity induced by electrical stimulation significantly increases anterograde and retrograde mitochondrial traffic in comparison with silent axons. Higher frequency conduction within a physiological range (50 Hz) dramatically further increased anterograde, but not retrograde, mitochondrial traffic, by rapidly increasing the number of mobile mitochondria and gradually increasing their velocity. Similarly, topical application of capsaicin to skin innervated by the saphenous nerve increased mitochondrial traffic in both myelinated and unmyelinated axons. In addition, stationary mitochondria in axons conducting at higher frequency become shorter, thus supplying additional mitochondria to the trafficking population, presumably through enhanced fission. Mitochondria recruited to the mobile population do not accumulate near Nodes of Ranvier, but continue to travel anterogradely. This pattern of mitochondrial redistribution suggests that the peripheral terminals of sensory axons represent sites of particularly high metabolic demand during physiological high frequency conduction. As the majority of mitochondrial biogenesis occurs at the cell body, increased anterograde mitochondrial traffic may represent a mechanism that ensures a uniform increase in mitochondrial density along the length of axons during high impulse load, supporting the increased metabolic demand imposed by sustained conduction.

Mesenchymal stem cells lack efficacy in the treatment of experimental autoimmune neuritis despite in vitro inhibition of T-cell proliferation.

Mesenchymal stem cells have been demonstrated to ameliorate experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, prompting clinical trials in multiple sclerosis which are currently ongoing. An important question is whether this therapeutic effect generalises to other autoimmune neurological diseases. We performed two trials of efficacy of MSCs in experimental autoimmune neuritis (EAN) in Lewis (LEW/Han (M)Hsd) rats, a model of human autoimmune inflammatory neuropathies. No differences between the groups were found in clinical, histological or electrophysiological outcome measures. This was despite the ability of mesenchymal stem cells to inhibit proliferation of CD4+ T-cells in vitro. Therefore the efficacy of MSCs observed in autoimmune CNS demyelination models do not necessarily generalise to the treatment of other forms of neurological autoimmunity.

The role of CD8(+) T cells in a model of multiple sclerosis induced with recombinant myelin oligodendrocyte glycoprotein.

BACKGROUND AND OBJECTIVES: Since CD8(+) T cells may be important in the pathogenesis of multiple sclerosis (MS), we examined their role in the DA rat experimental autoimmune encephalomyelitis (EAE) model induced by immunization with recombinant myelin oligodendrocyte glycoprotein (rMOG). METHODS: The inflammatory infiltrate in the spinal cord of affected animals was assessed by histology, electrophysiology and flow cytometry during the course of the disease (the first peak, remission and the second peak). The proportions of activated/memory effector (CD8(+)CD44(+)) and putative suppressor (CD8(+)CD28(-), CD8(+)CD25(high)) CD8(+) T cells in the draining lymph nodes were determined. To explore the role of CD8(+) T cells, similar experiments were performed in CD8(+) T cell depleted rats, before, during and after the first peak of the disease. RESULTS: Throughout the disease, both CD4(+) T cells and macrophages/activated microglia outnumbered CD8(+) T cells within the spinal cord. The number of putative suppressor CD8(+) T cells increased significantly both during and after the first peak suggesting the induction of a regulatory CD8(+) T-cell response. However, antibody-mediated depletion of CD8(+) T cells before induction of the disease, or after the first peak, did not significantly alter the incidence, severity or course of rMOG-induced EAE. CONCLUSIONS: The findings suggest that CD8(+) T cells do not play a significant role in the pathogenesis or regulation of EAE induced by rMOG in DA rats. In this respect, rMOG-induced EAE is not an appropriate model for studying the role of CD8(+) T cells in MS.

Delirium and the functional recovery of older medical inpatients after acute illness: the significance of biological factors.

Previous studies have not clarified the relationship of delirium to functional capacity during acute illness. We have investigated this relationship, incorporating the potential roles of APOE genotype and circulating cytokines in a longitudinal study of acutely admitted patients aged 70+ years. In all participants was measured the: Barthel Index (BI), mini-mental state examination (MMSE), confusion assessment method (CAM), delirium rating scale (DRS), APACHE II, APOE genotype. In a sub-sample: serum interferon-γ (IFN-γ), interleukin-1 (Levels of IL-1α, IL-1ß and IL-1 receptor antagonist activity IL-1RA), interleukin-6 (IL-6), leukemia inhibitory factor (LIF), tumor necrosis factor-α (TNF-α) and insulin-like growth factor-I (IGF-I). Of 164 participants, mean age 84.6 ± 6.57 years (± S.D.), 67.1% were women. On first assessment, mean BI was 14.13 ± 4.46 and delirium prevalence was 25.6%. At discharge, the mean BI of survivors (n=150) was 15.61 ± 4.22. By discharge, survivors who had recovered from prevalent delirium had significant improvement in BI (n=38, p=0.005), but non-recovers did not (n=14, p=0.512). On, multivariate analysis, BI was significantly affected by MMSE, APOE, IL-1α, IL-6, LIF and TNF-α levels (p<0.05) but not by delirium. Delirium in acutely admitted patients is associated with functional decline only in those who do not recover. Biological factors, rather that delirium itself, may be responsible for this.

Circulating subsets and CD4(+)CD25(+) regulatory T cell function in chronic inflammatory demyelinating polyradiculoneuropathy.

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an inflammatory disease of the peripheral nervous system that is probably autoimmune in origin. Different components of the adaptive and innate immunity may be responsible for the aberrant response towards nerve antigens. To investigate this, we examined lymphocyte subsets and regulatory T cell (Treg) function in the blood of CIDP patients, healthy controls (HC) and subjects with non-immune mediated neuropathies (other neuropathies, ON). We used flow cytometry to determine the frequency of monocytes, B cells, natural killer (NK) and NK-T cells, total and activated CD4(+) and CD8(+) T cells, effector memory and central memory CD4(+) and CD8(+) T cells, and CD4(+)CD25(high)Foxp3(+) Tregs. Treg function was studied after polyclonal stimulation and antigen specific stimulation with myelin protein peptides in CIDP and HC. There was an increased frequency of monocytes (p = 0.02) and decreased frequency of NK cells (p = 0.02) in CIDP compared with HC but not ON. There were no significant differences in other populations. Treg function was impaired in CIDP compared to HC (p = 0.02), whilst T cell proliferation to myelin protein peptides before and after depletion of Tregs was not different between patients and controls. This study shows increased circulating monocytes and reduced NK cells in CIDP. Although Treg frequency was not altered, we confirm that Tregs display a defect of suppressive function. Myelin protein peptides were not the target of the altered peripheral regulation of the immune response. The mechanisms of peripheral immune tolerance in CIDP and their relevance to the pathogenesis deserve further exploration.

Cytokines and IGF-I in delirious and non-delirious acutely ill older medical inpatients.

BACKGROUND: therapeutic use of cytokines can induce delirium, and delirium often occurs during infections associated with elevated levels of cytokines. This study examined the association of demographic, clinical and biological factors (IL-1alpha, IL-1beta, IL-1RA, IL-6, TNF-alpha, IFN-gamma, LIF, IGF-I, APOE genotype) with the presence and severity of delirium. METHODS: in an observational prospective longitudinal study, patients aged 70+ were recruited from an elderly medical unit and assessed every 3-4 days (maximum assessments 4). At each time, the scales MMSE, DRS, CAM, APACHEII were administered and blood was withdrawn to estimate the above biological factors. Mixed effects (PQL) and GEE were used to analyse the repeated measurements and investigate the associations at the individual and population average levels. RESULTS: a total of 205 observations on 67 individuals were analysed. Lower levels of IGF-I, and lower levels of circulating IL-1RA, are significantly (P < 0.05) associated with delirium, while the remaining of cytokines, severity of illness and possession of epsilon 4 allele had a non-significant effect. This has been shown by both statistical methods. Similarly lower levels of IGF-I, and high levels of IFN-gamma, are statistically significantly (P < 0.05) associated with higher DRS scores (more severe delirium). CONCLUSIONS: this study finds that (i) low levels of both neuroprotective factors (IGF-I, IL-1RA) are associated with delirium, (ii) high IFN-gamma and low IGF-I have significant effects on delirium severity and (iii) otherwise the pro-inflammatory cytokines studied, APOE genotype and severity of illness do not appear to be associated, in older medically ill patients, with either delirium or severity of it.

Associations of delirium with in-hospital and in 6-months mortality in elderly medical inpatients.

BACKGROUND: Studies on the association between mortality and delirium in older hospital inpatients have produced conflicting results. This insconsistency might be explained by case-mix differences in terms of clinical or underlying patho-physiological processes. For example, both albumin and C-reactive protein (CRP) have been reported as predictors of in-hospital mortality and interleukin-6 of longer-term mortality. METHODS: We used data from a longitudinal study of delirium to investigate the delirium-mortality relationship. A cohort of 164 patients, 70+ years were assessed within 3 days of acute hospital admission and hence twice weekly until hospital discharge, for the presence and severity of delirium and a range of clinical and laboratory measures, including initial albumin (n = 149), CRP (n = 76) and cytokine (n = 60) levels. In-hospital and 6-months mortality were determined from clinical records and telephone contact. RESULTS: During hospitalisation 14 (8.5%) patients died, 6 with delirium: mortality was not associated with delirium. At 6 months, 119 of 150 (77.3%) discharged patients were still alive, 21 (14.0%) dead, and 13 (8.7%) uncontactable. In bivariate analysis, 6-months mortality was associated with older age (P = 0.013), lower albumin (P = 0.001), higher CRP (P = 0.014) and higher interleukin-6 levels (P = 0.007), but not with presence or severity of in-hospital delirium. After controlling for other variables significant predictors (P < 0.05) for six-month mortality were initial MMSE, albumin, interferon-lambda and interleukin-6. CONCLUSIONS: The lack of demonstrable association between delirium and mortality may reflect inadequate statistical power in this study due to low numbers. These findings, however, highlight specific patho-physiological factors which may be important in the prognosis after delirium.

Botulinum toxin for detrusor overactivity and symptoms of overactive bladder: where we are now and where we are going.

Botulinum toxin (BTX) treatment for overactive bladder and detrusor overactivity is becoming increasingly recognized as an excellent therapeutic option for treating patients refractory to anticholinergic agents. Results from open-label studies have suggested that this therapy is effective in neurogenic and idiopathic detrusor overactivity, yet validating evidence from randomized, placebo-controlled trials has been unavailable. The exact mechanism of action of BTX in the bladder is controversial, although evidence suggests that apart from preventing the presynaptic release of acetylcholine from the parasympathetic innervation to the bladder, it might have an effect on sensory mechanisms. The latter hypothesis could in part explain its effect on symptoms such as urgency. The purpose of this Review is to present the results of randomized, placebo-controlled trials in which BTX treatment for detrusor overactivity was investigated. Also the evidence supporting its potential dual mechanism of action in the bladder will be considered. In addition, the various techniques of administration of BTX are discussed and avenues for further research suggested.

Characterization of a monoclonal antibody specific for human peripheral myelin protein 22 and its use in immunohistochemical studies of the fetal and adult nervous system.

We produced a mouse monoclonal antibody using cDNA and peptide immunization against the putative second extra-cellular domain of human peripheral myelin protein 22 (PMP22). It reacted specifically with human PMP22 and not with other human myelin proteins and did not react with bovine, rat, or mouse PMP22. The antibody stained the compact myelin of human peripheral nerve motor and sensory axons and did not stain central nervous system tissue. PMP22 reactivity was detected in the spinal roots of the human fetus at 19-20 weeks of gestation. The staining pattern of the PMP22 antibody resembled that of a monoclonal antibody directed against the myelin protein zero.

APOE and cytokines as biological markers for recovery of prevalent delirium in elderly medical inpatients.

BACKGROUND: Delirium frequently occurs in the context of infection and other inflammatory conditions associated with elevated levels of cytokines. Cytokines used therapeutically can induce symptoms of delirium as an adverse effect. We hypothesized that a causal relationship might exist between delirium and cytokine production during illness. Further, we speculated that the APOE genotype of patients might influence their rate of recovery from delirium given that APOE is associated with amyloid deposition, increased susceptibility to exogenous neurotoxins, and can affect the immune response. METHODS: A cohort of 164 acutely ill patients, 70 years or older, admitted to an elderly medical unit were studied within 3 days of hospital admission and re-assessed twice weekly until their discharge, to identify and follow the clinical course of delirium. The APOE genotype and the level of circulating cytokines were determined for 116 and 60 patients respectively. RESULTS: Prevalent delirium was significantly (p < 0.05) associated with a previous history of dementia, age, illness severity, disability and low levels of circulating IGF-I. Recovery was significantly associated (p < 0.05) with lack of APOE 4 allele and higher initial IFN-gamma. A model incorporating gender, APOE epsilon 4 status and IGF-I levels predicted recovery or not from delirium in 76.5% of cases, with a sensitivity 0.77 and specificity 0.75. CONCLUSIONS: A relationship between delirium with APOE genotype, IFN-gamma, and IGF-I, but not with IL-6, IL-1, TNF-alpha, and LIF was found. A predictive model of recovery was derived from gender, APOE status, and IGF-I levels. This model needs replication with further studies.

Reduced circulating CD4+CD25+ cell populations in Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is a monophasic inflammatory disease considered to be due to autoimmunity. In order to test the hypothesis that the disease is associated with a perturbation of the circulating lymphoid cell population, we tested the mononuclear cells from the venous blood of 21 patients with Guillain-Barré syndrome (GBS) and 20 healthy controls by flow cytometry. The proportions and numbers of B and T lymphocytes, and CD4, CD8, double negative and gammadelta T cell subsets and numbers of monocytes were not significantly different in the patients compared with the controls. However, the number and proportion of CD4+CD25+ cells were reduced in acute GBS (mean number 61.7 cells/microl, 95% CI 42.9-80.4 and mean percentage 4.6%, 95% CI 3.8-5.4) compared with controls (mean number 99.8 cells/microl, 95% CI 74.7-124.9, p=0.02, and mean percentage 6.0%, 95% CI 4.9-7.1%, p=0.037). In addition, in GBS patients, the number and proportion of CD4+ T cells expressing CD25+ and HLA-DP, DQ, DR (mean number 11.9 cells/microl, 95% CI 7.6-16.1 and mean percentage 0.8%, 95% CI 0.5-1.1%) was lower than in healthy controls (23.5 cells/microl, 95% CI 16.4-30.6, p=0.01, and mean percentage 1.4%, 95% CI 1.1-1.8%, p=0.005. Since CD4+CD25+ cells include cells with special immunoregulatory functions, further investigation of this phenomenon and its relation to possible loss of regulatory T cell function in GBS is warranted.

Pathogenesis of chronic inflammatory demyelinating polyradiculoneuropathy.

The acute lesions of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) consist of endoneurial foci of chemokine and chemokine receptor expression and T cell and macrophage activation. The myelin protein antigens, P2, P0, and PMP22, each induce experimental autoimmune neuritis in rodent models and might be autoantigens in CIDP. The strongest evidence incriminates P0, to which antibodies have been found in 20% of cases. Failure of regulatory T-cell mechanism is thought to underlie persistent or recurrent disease, differentiating CIDP from the acute inflammatory demyelinating polyradiculoneuropathy form of Guillain-Barré syndrome. Corticosteroids, intravenous immunoglobulin and plasma exchange each provide short term benefit but the possible long-term benefits of immunosuppressive drugs have yet to be confirmed in randomised, controlled trials.

Antibodies to peripheral nerve myelin proteins in chronic inflammatory demyelinating polyradiculoneuropathy.

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an acquired disorder of the peripheral nervous system with a probable auto-immune pathogenesis. The nature of the responsible autoantigens is unclear in most patients. We used the Western immunoblot technique to seek antibodies to peripheral nerve protein antigens. Sera from eight of 32 (25%) CIDP patients, 12 of 37 (32%) Guillain-Barré syndrome (GBS) patients, zero of 30 (0%) chronic idiopathic axonal polyneuropathy patients and two of 39 (5%) healthy control subjects contained anti-peripheral nerve protein antibodies. The frequency of such antibodies was significantly greater in both CIDP (p = 0.04) and GBS (p = 0.003) patients than in normal control subjects. For CIDP patients, there were non-significant trends for antibodies to be more common in females and in those who responded to treatment with either intravenous immunoglobulin or plasma exchange. The commonest antibodies were directed against a band at 28 kDa, resembling that labelled by a monoclonal antibody against myelin protein zero (P0). Six CIDP and seven GBS patients' sera reacted with this band. These results support the view that antibodies to myelin proteins, and especially P0, are present in the serum of some patients with CIDP and GBS.

Inflammation and primary demyelination induced by the intraspinal injection of lipopolysaccharide.

Inflammation is a prominent feature of several disorders characterized by primary demyelination, but it is not clear whether a relationship exists between inflammation and myelin damage. We have found that substantial demyelination results from the focal inflammatory lesion caused by the injection of lipopolysaccharide (LPS; 200 ng) directly into the rat dorsal funiculus. Within 24 h, such injections caused a focal inflammatory response consisting of a substantial number of polymorphonuclear cells and ED1-positive and inducible nitric oxide synthase (iNOS)-positive macrophages/microglia. The number of inflammatory cells was substantially reduced by day 7. OX-52-positive T-cells were less frequently observed but were present in the meninges at 8 h, reached a maximum in the dorsal funiculus at 7 days, and were rare at 14 days. The inflammation was followed by the appearance of a large lesion of primary demyelination that encompassed up to approximately 75% of the cross-sectional area of the dorsal funiculus. Treatment with dexamethasone significantly reduced the number of cells expressing iNOS, but did not prevent the demyelination. By 28 days the lesions were largely remyelinated, usually by Schwann cells. These changes were not observed in control, saline-injected animals. We conclude that the intraspinal injection of LPS results in inflammation and subsequently in prominent demyelination. The mechanisms underlying the demyelination are not clear, but it is notable that it typically begins with disruption of the adaxonal myelin. Indeed, there is an early loss of myelin-associated glycoprotein within the lesion, despite the persistence of proteolipid protein. This combination is a feature of the pattern III lesion recently described in multiple sclerosis (Lucchinetti et al., 2000), and we therefore suggest that LPS-induced demyelination may serve as the first experimental model available for the study of this type of multiple sclerosis lesion.

Axonal protection in experimental autoimmune neuritis by the sodium channel blocking agent flecainide.

Inflammatory demyelinating neuropathies such as Guillain-Barre syndrome (GBS) and its animal model, experimental autoimmune neuritis (EAN), are typically acute monophasic diseases of the PNS that can leave affected individuals with permanent disability due primarily to axonal degeneration. The mechanisms underlying the degeneration are not understood, but we have previously shown in vitro and in vivo that axons can degenerate when exposed to the inflammatory mediator nitric oxide, and that axons can be protected by application of the sodium channel-blocking agent, flecainide. Here we examine whether flecainide administration can similarly reduce axonal degeneration in the periphery in animals with EAN. EAN was induced in Lewis rats (n = 116, in three independent trials), and rats received either flecainide (Flec) (30 mg/kg/day) or vehicle (Veh) from the onset of disease expression. Flecainide administration significantly reduced the mean (SD) scores for neurological deficit at both the peak of disease (Flec: 5.7 (2.7), Veh: 8.0 (3.6), P < 0.001) and at the termination of the trials 25-29 days post-inoculation (Flec: 2.2 (2.4), Veh: 4.2 (4.2), P < 0.001). Histological examination of the tibial nerve of EAN animals revealed that flecainide provided significant protection against axonal degeneration so that 80.0% of the normal number of axons survived in flecainide-treated rats compared with 62.8% in vehicle-treated rats (P < 0.01). These findings may indicate a novel avenue for axonal protection in GBS and other inflammatory demyelinating neuropathies.

Brain-derived neurotrophic factor in experimental autoimmune neuritis.

Long-term disability in Guillain-Barré syndrome (GBS) is associated with axonal, and some neuronal, degeneration. Brain-derived neurotrophic factor (BDNF) can prevent neuronal death following damage to motor axons and we have therefore examined the ability of BDNF to ameliorate the effects of experimental autoimmune neuritis (EAN), a model of GBS. Treatment of Lewis rats with BDNF (10 mg/kg/day) did not significantly affect the neurological deficit, nor significantly improve survival, motor function or motor innervation. The weight of the urinary bladder was significantly increased in control animals with EAN, but remained similar to normal in animals treated with BDNF. With the exception of a possibly protective effect indicated by bladder weight, this study suggests that BDNF may not provide an effective therapy for GBS, at least in the acute phase of the disease.

A novel paralogous gene family involved in phase-variable flagella-mediated motility in Campylobacter jejuni.

Flagella-mediated motility is recognized to be one of the major factors contributing to virulence in Campylobacter jejuni. Motility of this bacterium is known to be phase variable, although the mechanism of such variation remains unknown. C. jejuni genome sequencing revealed a number of genes prone to phase variation via a slipped-strand mispairing mechanism. Many of these genes are hypothetical and are clustered in the regions involved in formation of three major cell surface structures: capsular polysaccharide, lipooligosaccharide and flagella. Among the genes of unknown function, the flagellar biosynthesis and modification region contains seven hypothetical paralogous genes designated as the motility accessory factor (maf) family. Remarkably, two of these genes (maf1 and maf4) were found to be identical and both contain homopolymeric G tracts. Using insertional mutagenesis it was demonstrated that one of the genes, maf5, is involved in formation of flagella. Phase variation of the maf1 gene via slipped-strand mispairing partially restored motility of the maf5 mutant. The maf family represents a new class of bacterial genes related to flagellar biosynthesis and phase variation. Reversible expression of flagella may be advantageous for the adaptation of C. jejunito the varied in vivo and ex vivo environments encountered during its life cycle, as well in evasion of the host immune response.