Lipocalin-2 as an Infection-Related Biomarker to Predict Clinical Outcome in Ischemic Stroke

Objectives From previous data in animal models of cerebral ischemia, lipocalin-2 (LCN2), a protein related to neutrophil function and cellular iron homeostasis, is supposed to have a value as a biomarker in ischemic stroke patients. Therefore, we examined LCN2 expression in the ischemic brain in an animal model and measured plasma levels of LCN2 in ischemic stroke patients. Methods In the mouse model of transient middle cerebral artery occlusion (tMCAO), LCN2 expression in the brain was analyzed by immunohistochemistry and correlated to cellular nonheme iron deposition up to 42 days after tMCAO. In human stroke patients, plasma levels of LCN2 were determined one week after ischemic stroke. In addition to established predictive parameters such as age, National Institutes of Health Stroke Scale and thrombolytic therapy, LCN2 was included into linear logistic regression modeling to predict clinical outcome at 90 days after stroke. Results Immunohistochemistry revealed expression of LCN2 in the mouse brain already at one day following tMCAO, and the amount of LCN2 subsequently increased with a maximum at 2 weeks after tMCAO. Accumulation of cellular nonheme iron was detectable one week post tMCAO and continued to increase. In ischemic stroke patients, higher plasma levels of LCN2 were associated with a worse clinical outcome at 90 days and with the occurrence of post-stroke infections. Conclusions LCN2 is expressed in the ischemic brain after temporary experimental ischemia and paralleled by the accumulation of cellular nonheme iron. Plasma levels of LCN2 measured in patients one week after ischemic stroke contribute to the prediction of clinical outcome at 90 days and reflect the systemic response to post-stroke infections

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

Anti-CD20 treatment effectively attenuates cortical pathology in a rat model of widespread cortical demyelination

Abstract Background Cortical demyelination represents a prominent feature of the multiple sclerosis (MS) brain, especially in (late) progressive stages. We recently developed a new rat model that reassembles critical features of cortical pathology characteristic to progressive types of MS. In persons affected by MS, B-cell depleting anti-CD20 therapy proved successful in the relapsing remitting as well as the early progressive course of MS, with respect to reducing the relapse rate and number of newly formed lesions. However, if the development of cortical pathology can be prevented or at least slowed down is still not clear. The main goal of this study was thus to increase our understanding for the mode of action of B-cells and B-cell directed therapy on cortical lesions in our rat model. Methods For this purpose, we set up two separate experiments, with two different induction modes of B-cell depletion. Brain tissues were analyzed thoroughly using histology. Results We observed a marked reduction of cortical demyelination, microglial activation, astrocytic reaction, and apoptotic cell loss in anti-CD20 antibody treated groups. At the same time, we noted increased neuronal preservation compared to control groups, indicating a favorable impact of anti-CD20 therapy. Conclusion These findings might pave the way for further research on the mode of action of B-cells and therefore help to improve therapeutic options for progressive MS

Sex Differences under Vitamin D Supplementation in an Animal Model of Progressive Multiple Sclerosis

A central role for vitamin D (VD) in immune modulation has recently been recognized linking VD insufficiency to autoimmune disorders that commonly exhibit sex-associated differences. Similar to other autoimmune diseases, there is a higher incidence of multiple sclerosis (MS) in women, but a poorer prognosis in men, often characterized by a more rapid progression. Although sex hormones are most likely involved, this phenomenon is still poorly understood. Oxidative stress, modulated by VD serum levels as well as sex hormones, may act as a contributing factor to demyelination and axonal damage in both MS and the corresponding preclinical models. In this study, we analyzed sex-associated differences and VD effects utilizing an animal model that recapitulates histopathological features of the progressive MS phase (PMS). In contrast to relapsing–remitting MS (RRMS), PMS has been poorly investigated in this context. Male (n = 50) and female (n = 46) Dark Agouti rats received either VD (400 IU per week; VD+) or standard rodent food without extra VD (VD−) from weaning onwards. Myelination, microglial activation, apoptotic cell death and neuronal viability were assessed using immunohistochemical markers in brain tissue. Additionally, we also used two different histological markers against oxidized lipids along with colorimetric methods to measure protective polyphenols (PP) and total antioxidative capacity (TAC) in serum. Neurofilament light chain serum levels (sNfL) were analyzed using single-molecule array (SIMOA) analysis. We found significant differences between female and male animals. Female rats exhibited a better TAC and higher amounts of PP. Additionally, females showed higher myelin preservation, lower microglial activation and better neuronal survival while showing more apoptotic cells than male rats. We even found a delay in reaching the peak of the disease in females. Overall, both sexes benefitted from VD supplementation, represented by significantly less cortical, neuroaxonal and oxidative damage. Unexpectedly, male rats had an even higher overall benefit, most likely due to differences in oxidative capacity and defense systems

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

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 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

Number of pups analysed with respect of developmental stage.

<p>E20 means day 20 of gestation, p2, p10, p15, p21, p32, p42 represents the respective age in days (postnatal days) at time of sacrifice.</p