Intrastriatal injection of interleukin-1 beta triggers the formation of neuromyelitis optica-like lesions in NMO-IgG seropositive rats

BACKGROUND: Neuromyelitis optica (NMO) is a severe, disabling disease of the central nervous system (CNS) characterized by the formation of astrocyte-destructive, neutrophil-dominated inflammatory lesions in the spinal cord and optic nerves. These lesions are initiated by the binding of pathogenic aquaporin 4 (AQP4)-specific autoantibodies to astrocytes and subsequent complement-mediated lysis of these cells. Typically, these lesions form in a setting of CNS inflammation, where the blood–brain barrier is open for the entry of antibodies and complement. However, it remained unclear to which extent pro-inflammatory cytokines and chemokines contribute to the formation of NMO lesions. To specifically address this question, we injected the cytokines interleukin-1 beta, tumor necrosis factor alpha, interleukin-6, interferon gamma and the chemokine CXCL2 into the striatum of NMO-IgG seropositive rats and analyzed the tissue 24 hours later by immunohistochemistry. RESULTS: All injected cytokines and chemokines led to profound leakage of immunoglobulins into the injected hemisphere, but only interleukin-1 beta induced the formation of perivascular, neutrophil-infiltrated lesions with AQP4 loss and complement-mediated astrocyte destruction distant from the needle tract. Treatment of rat brain endothelial cells with interleukin-1 beta, but not with any other cytokine or chemokine applied at the same concentration and over the same period of time, caused profound upregulation of granulocyte-recruiting and supporting molecules. Injection of interleukin-1 beta caused higher numbers of blood vessels with perivascular, cellular C1q reactivity than any other cytokine tested. Finally, the screening of a large sample of CNS lesions from NMO and multiple sclerosis patients revealed large numbers of interleukin-1 beta-reactive macrophages/activated microglial cells in active NMO lesions but not in MS lesions with comparable lesion activity and location. CONCLUSIONS: Our data strongly suggest that interleukin-1 beta released in NMO lesions and interleukin-1 beta-induced production/accumulation of complement factors (like C1q) facilitate neutrophil entry and BBB breakdown in the vicinity of NMO lesions, and might thus be an important secondary factor for lesion formation, possibly by paving the ground for rapid lesion growth and amplified immune cell recruitment to this site

Maternal neurofascin-specific autoantibodies bind to structures of the fetal nervous system during pregnancy, but have no long term effect on development in the rat

Neurofascin was recently reported as a target for axopathic autoantibodies in patients with multiple sclerosis (MS), a response that will exacerbate axonal pathology and disease severity in an animal model of multiple sclerosis. As transplacental transfer of maternal autoantibodies can permanently damage the developing nervous system we investigated whether intrauterine exposure to this neurofascin-specific response had any detrimental effect on white matter tract development. To address this question we intravenously injected pregnant rats with either a pathogenic anti-neurofascin monoclonal antibody or an appropriate isotype control on days 15 and 18 of pregnancy, respectively, to mimic the physiological concentration of maternal antibodies in the circulation of the fetus towards the end of pregnancy. Pups were monitored daily with respect to litter size, birth weight, growth and motor development. Histological studies were performed on E20 embryos and pups sacrificed on days 2, 10, 21, 32 and 45 days post partum. Results: Immunohistochemistry for light and confocal microscopy confirmed passively transferred anti-neurofascin antibody had crossed the placenta to bind to distinct structures in the developing cortex and cerebellum. However, this did not result in any significant differences in litter size, birth weight, or general physical development between litters from control mothers or those treated with the neurofascin-specific antibody. Histological analysis also failed to identify any neuronal or white matter tract abnormalities induced by the neurofascin-specific antibody. Conclusions: We show that transplacental transfer of circulating anti-neurofascin antibodies can occur and targets specific structures in the CNS of the developing fetus. However, this did not result in any pre- or post-natal abnormalities in the offspring of the treated mothers. These results assure that even if anti-neurofascin responses are detected in pregnant women with multiple sclerosis these are unlikely to have a negative effect on their children

Exploring the attitude of Faculty of Medicine students about herbal products usage: a cross-sectional study

Herbal products have gained popularity, and they are often perceived as safe. The roles of the pharmacists and physicians are essential in the proper usage of those products. This study aimed to investigate the students of Faculty of Medicine in Niš about the use of herbal preparations and the differences between medicine, pharmacy and dentistry students. The fifth-year university students were questioned through an anonymous survey. The obtained results were processed statistically and presented graphically. The majority of the students would prefer a combination of herb product and drug (76.9%). Usage of herbal products is based on students' knowledge, on the advice of pharmacists, family members, physicians, and the media. The majority of students purchase herbal products in the pharmacy and nobody obtained them via the Internet. Most of the students used them in preventive purposes (60%) and claimed that there were positive effects. Students (75%) read the instructions for use and side effects, especially pharmacy students (88.3%). Medicine students (60%) consider herbal products completely safe. Pharmacy students are considered to receive the necessary knowledge on herbal products (68.3%), while a smaller percentage of medicine and dentistry students consider the same. Students use herbal medicines but are not entirely familiar with their effects and safety. There is a need for raising awareness, the importance of side effects and interactions of herbs and drugs among medicine and dentistry students. This goal can be achieved by changing curriculum which would contain material from phytotherapy

Pathogenic T cell responses against aquaporin 4

Inflammatory lesions in the central nervous system of patients with neuromyelitis optica are characterized by infiltration of T cells and deposition of aquaporin-4-specific antibodies and complement on astrocytes at the glia limitans. Although the contribution of aquaporin-4-specific autoantibodies to the disease process has been recently elucidated, a potential role of aquaporin-4-specific T cells in lesion formation is unresolved. To address this issue, we raised aquaporin-4-specific T cell lines in Lewis rats and characterized their pathogenic potential in the presence and absence of aquaporin-4-specific autoantibodies of neuromyelitis optica patients. We show that aquaporin-4-specific T cells induce brain inflammation with particular targeting of the astrocytic glia limitans and permit the entry of pathogenic anti-aquaporin-4-specific antibodies to induce NMO-like lesions in spinal cord and brain. In addition, transfer of aquaporin-4-specific T cells provoked mild (subclinical) myositis and interstitial nephritis. We further show that the expression of the conformational epitope, recognized by NMO patient-derived aquaporin-4-specific antibodies is induced in kidney cells by the pro-inflammatory cytokine gamma-interferon. Our data provide further support for the view that NMO lesions may be induced by a complex interplay of T cell mediated and humoral immune responses against aquaporin-4

The role of proinflammatory cytokines in the pathogenesis of neuromyelitis optica

Neuromyelitis optica (NMO) ist eine schwere, demyelinisierende Erkrankung des Zentralnervensystems (ZNS), welche vor Allem den optischen Nerven und das Rückenmark betrifft. Im Verlauf der Erkrankung können diese Beeinträchtigungen bis zur Erblindung und vollständigen Lähmung führen. Trotz vieler neuer Erkenntnisse in den letzten Jahrzehnten, wie zum Beispiel der Entdeckung von krankheitsspezifischen Antikörpern, ist die genaue Pathogenese der Erkrankung noch immer ungeklärt, insbesondere Mechanismen die zu Beginn an der Auslösung der Erkrankung beteiligt sind. In dieser Arbeit haben wir untersucht, wie proinflammatorische Zytokine zur Entstehung von NMO ähnlichen Läsionen im ZNS von Lewis Ratten beitragen. Dazu haben wir proinflammatorische Schlüsselzytokine und Chemokine in das Striatum der Tiere injiziert. Zusätzlich erhielten die Tiere intraperitoneale Injektionen von Autoantikörpern (NMO-IgG), welche aus Patienten Serum gewonnen wurden oder humanes Kontroll-IgG. Anschließend haben wir mit Hilfe immunhistochemischer Methoden das ZNS dieser Tiere untersucht und konnten feststellen, dass Interleukin -1 beta (IL-1 beta) kleine, NMO ähnliche Läsionen im Kortex, Striatum und Thalamus hervorruft jedoch andere Mediatoren keinerlei signifikante Veränderungen auslösten. Die von IL-1 beta induzierten Läsionen waren gekennzeichnet durch Infiltration von Granulozyten und der Aktivierung von Mikroglia/Makrophagen. Zusätzlich kam es zu variabel ausgeprägter Zerstörung von Astrozyten und verschieden starker Ablagerung von Komplement in den betroffenen Regionen. Außerdem haben wir die Wirkung von proinflammtorischen Zytokinen auf primäre Endothelzellkulturen untersucht und konnten eine deutliche Hochregulierung von CXC Chemokinen und Adhäsionsmolekülen, welche in der Rekrutierung von Granulozyten beteiligt sind, im Zusammenhang mit der Zugabe von IL-1 beta beobachten. Die von uns erhobenen Daten deuten darauf hin, dass IL-1 beta ein wichtiges Zytokin ist, welches zur Bildung von Läsionen in NMO-IgG seropositiven Patienten beiträgt. Unterstützend zu diesen Daten konnten wir zeigen, dass die Konzentration von IL-1 beta in aktiven Läsionen von NMO-Patienten erhöht ist. Im Gegensatz dazu konnten wir keine erhöhten Werte bei vergleichbaren Läsionen von MS Patienten oder Kontrollgeweben finden.Neuromyelitis optica (NMO) is a severe, demyelinating disease of the central nervous system (CNS), affecting predominantly optic nerves and the spinal cord, which can eventually result in blindness and paralysis. Despite the tremendous progress that has been done over the last decade (e.g. the discovery of the disease-specific antibody), many aspects of the disease pathogenesis remain elusive, especially its initial events. In the present study, we analyzed the contribution of proinflammatory cytokines in the formation of NMO-like lesions in the CNS of Lewis rats. We injected key proinflammatory cytokines/ chemokines into the striatum of animals that additionally received intraperitoneal injections of patients' autoantibodies (NMO-IgG) or human control antibodies. We then studied the CNS of these animals in detail by immunohistochemistry and observed that interleukin-1 beta (IL-1 beta) induced small NMO-like lesions in the cortex, striatum and thalamus, while the effects of other mediators were insignificant. The IL-1 beta-induced lesions were characterized by granulocyte cell infiltration and microglia/ macrophage activation, and exhibited a variable degree of astrocyte destruction and complement deposition. In addition, we studied the effects of proinflammatory cytokines on primary rat brain endothelial cell cultures and observed a marked IL-1 beta-dependent upregulation of CXC chemokines and adhesion molecules, involved in granulocyte recruitment. All these data strongly suggest that IL-1 beta is an important cytokine contributing to the lesion formation in NMO-IgG seropositive hosts. Additionally, we found that active lesions of NMO patients displayed enhanced levels of IL-1 beta, a feature not observed in the CNS tissue derived from MS patients with comparable disease activity or in the control CNS tissue.submitted by Maja KiticAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheActa Neuropathologica Communications, 2013,1(1):5Wien, Med. Univ., Diss., 2013OeBB(VLID)171501

Microglia pre-activation and neurodegeneration precipitate neuroinflammation without exacerbating tissue injury in experimental autoimmune encephalomyelitis

Abstract Human inflammatory or neurodegenerative diseases, such as progressive multiple sclerosis (MS), occur on a background of age-related microglia activation and iron accumulation as well as pre-existing neurodegeneration. Most experimental models for CNS diseases, however, are induced in rodents, which are naturally characterized by a homeostatic microglia phenotype, low cellular iron load and absence of neurodegeneration. Here, we show that naïve LEWzizi rats – Lewis rats with a zitter rat background – show a spontaneous phenotype partly mimicking the changes seen in human aging and particularly in the normal-appearing white and grey matter of patients with progressive MS. Using this model system, we further aimed to investigate (i) whether the acute monophasic MS model experimental autoimmune encephalomyelitis (EAE) transforms into chronic progressive disease and (ii) whether EAE-induced neuroinflammation and tissue damage aggravate on the LEWzizi background. We found that the pre-existing LEWzizi-specific pathology precipitated EAE-related neuroinflammation into forebrain areas, which are devoid of EAE lesions in normal Lewis rats. However, EAE-related tissue damage was neither modified by the LEWzizi-specific pathology nor did EAE-induced neuroinflammation modify the LEWzizi-related pathological process. Our data indicate that the interaction between pre-activated microglia and CD4+ autoreactive T cells during the induction and propagation of tissue damage in the CNS is limited

Neurofascin antibodies pass the placental border and reach fetal tissues.

<p>Histological analysis of pups (developmental stage E20) 48 hours after application of the second dose of passive antibody transfer. Immunohistochemistry for mouse-immunoglobulin proves antibody transfer into fetal tissue. (a) shows mouse immunoglobulin reactivity in brain tissue vessels of the unborn rat pups, (b) in fetal liver, (c) in the kidney and (d) in the lung of the pups (original magnification 200×). Scale bars represent 50 µm.</p

Graphical presentation of vital statistics of neurofascin treated pups vs. control groups.

<p>The weight curve from age p2 up to p32 of neurofascin- and control antibody treated pups in comparison to an untreated control rat litter is shown in Fig. 4 a. Weight curves are remarkably similar with no significant differences noted. Also, the litter sizes did not differ significantly between the neurofascin antibody group and the control antibody group (<i>p</i> = 0,336712), as shown in Fig. 4 b, ruling out significant fetal loss due to the presence of the antiaxonal antibody. Fig. 4c shows the mean times in seconds +− SD on an accelerating Rotarod device tested on postnatal days 16, 19, 24, 25 and 32. Again, no statistical significant difference between the groups was found (untreated control vs. control antibody group <i>p</i> = 0,530163, untreated control vs. neurofascin treatment <i>p</i> = 0,354292, control antibody group vs. neurofascin group <i>p</i> = 0,117562). Fore limb grip test was performed on postnatal days 24, 25 and 32 and is shown in Fig. 4d. Again, no statistically significant differences were noted (untreated control vs. control antibody group <i>p</i> = 0,935596, untreated control vs. neurofascin treatment <i>p</i> = 0,429921, control antibody group vs. neurofascin group <i>p</i> = 0,593029).</p

Neurofascin immunoreactivity is not altered by intrauterine exposure to anti- neurofascin autoantibodies.

<p>Immunohistochemistry for neurofascin in the normal adult rat brain (a-d) and at different stages of development after intrauterine exposure to the anti- neurofascin antibody (e-i). Areas marked with a rectangle in Fig. 2a and b are shown at higher magnification in c and d, respectively. (a, c) shows immunoreactivity for neurofascin in axons in the hippocampus region, (b, d) in purkinje cells of the cerebellum of normal, untreated adult rats. (e) shows neurofascin staining in the hippocampus region in rats at E20, (f, g) in purkinje cells at postnatal day 10, (h) in purkinje cells at p 21 and (i) in axons of the hippocampus at p21. The arrows in 2d, f-h show the specific neurofascin immunoreactivity of the initial axonal segments of the purkinje cells. (original magnification in a, b 100×, in c-i 630×). Scale bars in a, b represents 100 µm, in c-i 20 µm.</p

Neurofascin antibodies bind to their target structures in the fetal rat brain.

<p>Double stainings for light microscopy for neurofascin (brown) and mouse IgG (red) in rats at p21 are shown in Fig. 3 a-d. In neurofascin antibody treated rats (3a, c) an intense double staining for neurofascin as well as mouse Ig is detectable in axons of the hippocampus region (a) and purkinje cells (c), proving the mouse- derived anti-neurofascin antibody bound to its target structures. Animals treated with control antibody (3b, d) show reactivity for neurofascin in the respective areas, but no staining for mouse immunoglobulin. The black arrows in 3 c, d show the initial axonal segments of the purkinje cells. This staining pattern is confirmed by immunohistochemistry for confocal microscopy of the hippocampus region; staining pattern for neurofascin (e, g) is identical in neurofascin antibody treated animals (e) and control animals (g), but mouse immunoglobulin is only detectable in the neurofascin group (f) but not in the control group (h). Scale bars in a-d represent 20 µm, and in e-h 100 µm.</p