[show abstract][hide abstract] ABSTRACT: Tumour necrosis factor (TNF) is a proinflammatory cytokine that is known to regulate inflammation in a number of autoimmune diseases, including multiple sclerosis (MS). Although targeting of TNF in models of MS has been successful, the pathological role of TNF in MS remains unclear due to clinical trials where the non-selective inhibition of TNF resulted in exacerbated disease. Subsequent experiments have indicated that this may have resulted from the divergent effects of the two TNF receptors, TNFR1 and TNFR2. Here we show that the selective targeting of TNFR1 with an antagonistic antibody ameliorates symptoms of the most common animal model of MS, experimental autoimmune encephalomyelitis (EAE), when given following both a prophylactic and therapeutic treatment regime. Our results demonstrate that antagonistic TNFR1-specific antibodies may represent a therapeutic approach for the treatment of MS in the future.
PLoS ONE 01/2014; 9(2):e90117. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Neurodegeneration has been increasingly recognised as the leading structural correlate of disability progression in autoimmune diseases such as multiple sclerosis. Since calcium signalling is known to regulate the development of degenerative processes in many cell types, it is believed to play significant roles in mediating neurodegeneration. Because of its function as a major juncture linking various insults and injuries associated with inflammatory attack on neuronal cell bodies and axons, it provides potential for the development of neuroprotective strategies. This is of great significance because of the lack of neuroprotective agents presently available to supplement the current array of immunomodulatory treatments. In this review, we summarise the role that various calcium channels and pumps have been shown to play in the development of neurodegeneration under inflammatory autoimmune conditions. The identification of suitable targets might also provide insights into applications in non-inflammatory neurodegenerative diseases.
Cell and Tissue Research 12/2013; · 3.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Optic neuritis is a common manifestation of multiple sclerosis, an inflammatory demyelinating disease of the CNS. Recently, the neurodegenerative component of multiple sclerosis has come under focus particularly because permanent disability in patients correlates well with neurodegeneration; and observations in both humans and multiple sclerosis animal models highlight neurodegeneration of retinal ganglion cells as an early event. After myelin oligodendrocyte glycoprotein immunization of Brown Norway rats, significant retinal ganglion cell loss precedes the onset of pathologically defined autoimmune optic neuritis. To study the role calcium and calpain activation may play in mediating early degeneration, manganese-enhanced magnetic resonance imaging was used to monitor preclinical calcium elevations in the retina and optic nerve of myelin oligodendrocyte glycoprotein-immunized Brown Norway rats. Calcium elevation correlated with an increase in calpain activation during the induction phase of optic neuritis, as revealed by increased calpain-specific cleavage of spectrin. The relevance of early calpain activation to neurodegeneration during disease induction was addressed by performing treatment studies with the calpain inhibitor calpeptin. Treatment not only reduced calpain activity but also protected retinal ganglion cells from preclinical degeneration. These data indicate that elevation of retinal calcium levels and calpain activation are early events in autoimmune optic neuritis, providing a potential therapeutic target for neuroprotection.
Journal of neuropathology and experimental neurology. 07/2013;
[show abstract][hide abstract] ABSTRACT: The ability to monitor changes in membrane potential is a useful tool for studying neuronal function, but there are only limited options available at present. Here, we have investigated the potential of a commercially available FLIPR membrane potential (FMP) dye, developed originally for high throughput screening using a plate reader, for imaging the membrane potential of cultured cells using an epifluorescence-based single cell imaging system. We found that the properties of the FMP dye make it highly suitable for such imaging since 1) its fluorescence displayed a high signal-to-noise ratio, 2) robust signals meant only minimal exposure times of around 5 ms were necessary, and 3) bidirectional changes in fluorescence were detectable resulting from hyper- or depolarising conditions, reaching equilibrium with a time constant of 4-8 s. Measurements were possible independently of whether membrane potential changes were induced by voltage clamping, or manipulating the ionic distribution of either Na(+) or K(+). Since FMP behaves as a charged molecule which accumulates in the cytosol, equations based on the Boltzmann distribution were developed determining that the apparent charge of FMP which represents a measure of the voltage sensitivity of the dye, is between -0.62 and -0.72. Finally, we demonstrated that FMP is suitable for use in a variety of neuronal cell types and detects membrane potential changes arising from spontaneous firing of action potentials and through stimulation with a variety of excitatory and inhibitory neurotransmitters.
PLoS ONE 01/2013; 8(3):e58260. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Neurodegeneration plays a major role in multiple sclerosis (MS), in which it is thought to be the main determinant of permanent disability. However, the relationship between the immune response and the onset of neurodegeneration is still a matter of debate. Moreover, recent findings in MS patients raised the question of whether primary neurodegenerative changes can occur in the retina independent of optic nerve inflammation. Using a rat model of MS that frequently leads to optic neuritis, we have investigated the interconnection between neurodegenerative and inflammatory changes in the retina and the optic nerves with special focus on preclinical disease stages. We report that, before manifestation of optic neuritis, characterized by inflammatory infiltration and demyelination of the optic nerve, degeneration of retinal ganglion cell bodies had already begun and ultrastructural signs of axon degeneration could be detected. In addition, we observed an early activation of resident microglia in the retina. In the optic nerve, the highest density of activated microglia was found within the optic nerve head. In parallel, localized breakdown in the integrity of the blood-retinal barrier and aberrations in the organization of the blood-brain barrier marker aquaporin-4 in the optic nerves were observed during the preclinical phase, before onset of optic neuritis. From these findings, we conclude that early and subtle inflammatory changes in the retina and/or the optic nerve head reminiscent of those suggested for preclinical MS lesions may initiate the process of neurodegeneration in the retina before major histopathological signs of MS become manifest.
Journal of Neuroscience 04/2012; 32(16):5585-97. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mice overexpressing proteolipid protein (PLP) develop a leukodystrophy-like disease involving cytotoxic, CD8+ T-lymphocytes. Here we show that these cytotoxic T-lymphocytes perturb retrograde axonal transport. Using fluorogold stereotactically injected into the colliculus superior, we found that PLP overexpression in oligodendrocytes led to significantly reduced retrograde axonal transport in retina ganglion cell axons. We also observed an accumulation of mitochondria in the juxtaparanodal axonal swellings, indicative for a disturbed axonal transport. PLP overexpression in the absence of T-lymphocytes rescued retrograde axonal transport defects and abolished axonal swellings. Bone marrow transfer from wildtype mice, but not from perforin- or granzyme B-deficient mutants, into lymphocyte-deficient PLP mutant mice led again to impaired axonal transport and the formation of axonal swellings, which are predominantly located at the juxtaparanodal region. This demonstrates that the adaptive immune system, including cytotoxic T-lymphocytes which release perforin and granzyme B, are necessary to perturb axonal integrity in the PLP-transgenic disease model. Based on our observations, so far not attended molecular and cellular players belonging to the immune system should be considered to understand pathogenesis in inherited myelin disorders with progressive axonal damage.
PLoS ONE 01/2012; 7(8):e42554. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although the pathologic role of the prion protein in transmissible spongiform encephalopathic diseases has been widely investigated, the physiologic role of the cellular prion protein (PrP(C)) is not known. Among the many functions attributed to PrP(C), there is increasing evidence that it is involved in cell survival and mediates neuroprotection. A potential role in the immune response has also been suggested. However, how these two functions interplay in autoimmune disease is unclear. To address this, autoimmune optic neuritis, a model of multiple sclerosis, was induced in C57Bl/6 mice, and up-regulation of PrP(C) was observed throughout the disease course. In addition, compared with wild-type mice, in PrP(C)-deficient mice and mice overexpressing PrP(C), histopathologic analysis demonstrated that optic neuritis was exacerbated, as indicated by axonal degeneration, inflammatory infiltration, and demyelination. However, significant neuroprotection of retinal ganglion cells, the axons of which form the optic nerve, was observed in mice that overexpressed PrP(C). Conversely, mice lacking PrP(C) demonstrated significantly more neurodegeneration. This suggests that PrP(C) may have a neuroprotective function independent of its role in regulating the immune response.
American Journal Of Pathology 06/2011; 178(6):2823-31. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Optical coherence tomography (OCT) is becoming the state-of-the-art method for the non-invasive imaging of a variety of ocular diseases. The aim of this study was to assess the application of OCT for the in vivo monitoring and follow-up of pathological changes during experimental autoimmune uveoretinitis (EAU) in rats. Initially we established OCT imaging in healthy brown Norway rats and correlated it with retinal histology. Subsequently, we induced EAU and imaged animals by OCT throughout the pre-peak, peak, and post-peak phases of the disease. The sensitivity of OCT imaging was determined by comparison with clinical EAU and histopathology scores obtained ex vivo at several time points throughout the disease course. Our data demonstrate that OCT imaging of the healthy rat retina closely correlates with histological observations and allows the clear visualization of all retinal layers. After induction of EAU, the first pathological changes could be detected by OCT at day (d) 8 post-immunization (p.i.) which corresponded to the time point of clinical disease onset. An increase in retinal thickness (RT) was detected from d10 p.i. onwards which peaked at d16 p.i. and decreased again to near control levels by d20 p.i. We introduce a novel semi-quantitative OCT scoring which correlates with histopathological findings and complements the clinical scores. Therefore, we conclude that OCT is an easily accessible, non-invasive tool for detection and follow-up of histopathological changes during EAU in rats. Indeed, significant differences in RT between different stages of EAU suggest that this OCT parameter is a sensitive marker for distinguishing disease phases in vivo.
Experimental Eye Research 05/2011; 93(1):82-90. · 3.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of this study was to investigate the role of voltage-dependent calcium channels (VDCCs) in axon degeneration during autoimmune optic neuritis.
Calcium ion (Ca(2+)) influx into the optic nerve (ON) through VDCCs was investigated in a rat model of optic neuritis using manganese-enhanced magnetic resonance imaging and in vivo calcium imaging. After having identified the most relevant channel subtype (N-type VDCCs), we correlated immunohistochemistry of channel expression with ON histopathology. In the confirmatory part of this work, we performed a treatment study using omega-conotoxin GVIA, an N-type specific blocker.
We observed that pathological Ca(2+) influx into ONs during optic neuritis is mediated via N-type VDCCs. By analyzing the expression of VDCCs in the inflamed ONs, we detected an upregulation of alpha(1B), the pore-forming subunit of N-type VDCCs, in demyelinated axons. However, high expression levels were also found on macrophages/activated microglia, and lower levels were detected on astrocytes. The relevance of N-type VDCCs for inflammation-induced axonal degeneration and the severity of optic neuritis was corroborated by treatment with omega-conotoxin GVIA. This blocker led to decreased axon and myelin degeneration in the ONs together with a reduced number of macrophages/activated microglia. These protective effects were confirmed by analyzing the spinal cords of the same animals.
We conclude that N-type VDCCs play an important role in inflammation-induced axon degeneration via two mechanisms: First, they directly mediate toxic Ca(2+) influx into the axons; and second, they contribute to macrophage/microglia function, thereby promoting secondary axonal damage. Ann Neurol 2009;66:81-93.
Annals of Neurology 04/2009; 66(1):81-93. · 11.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system that results in persistent impairment in young adults. During chronic progressive disease stages, there is a strong correlation between neurodegeneration and disability. Current therapies fail to prevent progression of neurological impairment during these disease stages. Flupirtine, a drug approved for oral use in patients suffering from chronic pain, was used in a rat model of autoimmune optic neuritis and significantly increased the survival of retinal ganglion cells, the neurons that form the axons of the optic nerve. When flupirtine was combined with interferon-beta, an established immunomodulatory therapy for MS, visual functions of the animals were improved during the acute phase of optic neuritis. Furthermore, flupirtine protected retinal ganglion cells from degeneration in a noninflammatory animal model of optic nerve transection. Although flupirtine was shown previously to increase neuronal survival by Bcl-2 up-regulation, this mechanism does not appear to play a role in flupirtine-mediated protection of retinal ganglion cells either in vitro or in vivo. Instead, we showed through patch-clamp investigations that the activation of inwardly rectifying potassium channels is involved in flupirtine-mediated neuroprotection. Considering the few side effects reported in patients who receive long-term flupirtine treatment for chronic pain, our results indicate that this drug is an interesting candidate for further evaluation of its neuroprotective potential in MS.
American Journal Of Pathology 11/2008; 173(5):1496-507. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: To assess the use of visual evoked potentials (VEPs) for the in vivo detection of impaired visual function in a marmoset model of multiple sclerosis. The sensitivity of the VEP recordings was determined by comparison with magnetic resonance imaging (MRI) and histopathology.
Baseline VEPs were recorded in six healthy marmoset monkeys in response to light-flash stimulation. Experimental autoimmune encephalomyelitis (EAE) was induced in four of the six monkeys. Clinical scores were assessed daily, and VEPs were recorded every second week. In vivo MRI and subsequent histopathology of the brains and optic nerves were performed at the end of the study.
After induction of EAE, all four marmosets exhibited clinical signs between day 26 and 38 after immunization. VEPs were normal during the induction phase of the disease, but deteriorated in amplitude with the occurrence of clinical symptoms in all animals. MRI revealed bilateral optic neuritis and signal alterations in the optic tracts and occipital subcortical white matter in two of the animals. In the remaining two animals, MRI detected signal alterations in the occipital subcortical white matter. Histopathologic results were concordant with the MRI findings.
VEPs are an easily accessible noninvasive tool for measuring visual function and diagnosing impairment of the visual pathway in a marmoset EAE model.
[show abstract][hide abstract] ABSTRACT: Axonal destruction and neuronal loss occur early during multiple sclerosis, an autoimmune inflammatory CNS disease that frequently manifests with acute optic neuritis. Available therapies mainly target the inflammatory component of the disease but fail to prevent neurodegeneration. To investigate the effect of minocycline on the survival of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve, we used a rat model of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis. Optic neuritis in this model was diagnosed by recording visual evoked potentials and RGC function was monitored by measuring electroretinograms. Functional and histopathological data of RGCs and optic nerves revealed neuronal and axonal protection when minocycline treatment was started on the day of immunization. Furthermore, we demonstrate that minocycline-induced neuroprotection is related to a direct antagonism of multiple mechanisms leading to neuronal cell death such as the induction of anti-apoptotic intracellular signalling pathways and a decrease in glutamate excitotoxicity. From these observations, we conclude that minocycline exerts neuroprotective effects independent of its anti-inflammatory properties. This hypothesis was confirmed in a non-inflammatory disease model leading to degeneration of RGCs, the surgical transection of the optic nerve.
Neurobiology of Disease 04/2007; 25(3):514-25. · 5.62 Impact Factor
[show abstract][hide abstract] ABSTRACT: Axonal destruction and neuronal loss occur early during multiple sclerosis (MS), an autoimmune inflammatory central nervous system disease that frequently manifests with acute optic neuritis. Glatiramer acetate (GA) and interferon-beta-1b (IFN-beta-1b) are two immunomodulatory agents that have been shown to decrease the frequency of MS relapses. However, the question of whether these substances can slow neurodegeneration in MS patients is the subject of controversy. In a rat model of experimental autoimmune encephalomyelitis, we investigated the effects of GA and IFN-beta-1b on the survival of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve. For each substance, therapy was started 14 days before immunization, on the day of immunization, or on the day of clinical disease onset. After myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis became clinically manifest, optic neuritis was monitored by recording visual evoked potentials. The function of RGCs was measured by electroretinograms. Although early GA or IFN-beta-1b treatment showed benefit on disease activity, only treatment with GA exerted protective effects on RGCs, as revealed by measuring neurodegeneration and neuronal function. Furthermore, we demonstrate that this GA-induced neuroprotection does not exclusively depend on the reduction of inflammatory infiltrates within the optic nerve.
American Journal Of Pathology 11/2006; 169(4):1353-64. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Interferon-beta-1a (IFN-beta-1a) is an approved treatment for multiple sclerosis (MS). It improves the disease course by reducing the relapse rate as well as the persistent neurological deficits. Recent MRI and post-mortem studies revealed that neuronal and axonal damage are most relevant for chronic disability in MS patients. We have characterized previously time course and mechanisms of neuronal apoptosis in a rat model of myelin oligodendrocyte glycoprotein (MOG)-induced optic neuritis. In this animal model, application of IFN-beta-1a three times per week slightly decreases the loss of retinal ganglion cells (RGCs), the neurons that form the axons within the optic nerve. In contrast to neurotrophic factors, this cytokine does not directly protect cultured RGCs from apoptosis. We conclude that IFN-beta-1a is a suitable candidate to be combined with a directly neuroprotective agent in order to further decrease axonal and neuronal degeneration in MS patients.