The chemokine fractalkine (CX3CL1) attenuates H2O2-induced demyelination in cerebellar slices

Abstract Background Fractalkine/CX3CR1 signalling has been implicated in many neurodegenerative and neurological diseases of the central nervous system (CNS). This signalling pathway plays an important role in regulating reactive oxygen species (ROS), as well as itself being altered in conditions of oxidative stress. Here, we investigated the effects of recombinant fractalkine (rCX3CL1) in models of hydrogen peroxide (H2O2)-induced demyelination and astrocyte toxicity, within organotypic cerebellar slice cultures. Methods Organotypic cerebellar slice cultures were generated from postnatal day 10 C57BL/6J mice to assess myelination. Immunohistochemistry was used to measure the degree of myelination. Fluorescent images were obtained using a leica SP8 confocal microscope and data analysed using ImageJ software. Results We show here, for the first time, that rCX3CL1 significantly attenuated bolus H2O2-induced demyelination as measured by expression of myelin basic protein (MBP) and attenuated reduced vimentin expression. Using the GOX-CAT system to continuously generate low levels of H2O2 and induce demyelination, we observed similar protective effects of rCX3CL1 on MBP and MOG fluorescence, although in this model, the decrease in vimentin expression was not altered. Conclusions This data indicates possible protective effects of fractalkine signalling in oxidative stress-induced demyelination in the central nervous system. This opens up the possibility of fractalkine receptor (CX3CR1) modulation as a potential new target for protecting against oxidative stress-induced demyelination in both inflammatory and non-inflammatory nervous system disorders

Attitudes and beliefs of parents of children with disabilities in Uganda

Background. Little is known about the experience of carers of children with disabilities in Uganda, where child disability constitutes 31.4% of all disabilities. This study examined the experiences, beliefs, and attitudes of parents/ main carers of children with disabilities, and the challenges they face, in order to optimize rehabilitation strategies for the child and their family. Methods. Qualitative data were collected from ten semi - structured interviews with eight main carers children receiving rehabilitation in rural Uganda. Results. Three main themes were identified: (1) experiences, (2) beliefs, and (3) attitudes of the parents/ main carers. Carers experienced emotional stress and many life changes as the burden of care fell primarily on them. A lack of knowledge and information about disability amongst carers, resulted in alternative beliefs about treatment. Social stigma towards disability remains an issue within Ugandan society. Conclusions. Family centered rehabilitation should be incorporated into rehabilitation programmes to decrease burden of care upon the main carer. Health care practitioners are in a strong position to educate families about causation, diagnosis, and prognosis of a child’s condition, but such services should be improved through community education about disability and facilitated with the development of parental support groups

ROS production or extracellular mediators are not involved in psychosine-induced toxicity in human astrocytes.

<p>(A) Human astrocytes were pre-treated with a specific ROS inhibitor N-acetylcysteine (NAC) (1mM for 1h) (n = 3–4) before treatment with psychosine (10μM for 3h or 20h). The main graph shows the ratio between control vs psychosine and drug vs drug+psychosine and smaller graphs show comparisons to vehicle control and psychosine treatment. (B) Human astrocytes were serum starved for 3h before treated with psychosine (10μM for 1h). After washing three times with serum free media to wash out the added psychosine, fresh complete media was incubated for 5h with the treated astrocytes and this conditioned media (“psy-media”) was then added to naive human astrocytes and incubated for 20h. One-way ANOVA and Newman-Keuls Multiple Comparison post-test, compared to control (*p<0.05, **p<0.01, ***p<0.005). The number of experiments is indicated in the parenthesis (n = 4–28). Cell viability was quantified by an MTT assay. Data is presented as mean + s.e.m.</p

DEDA treatment inhibits psychosine-induced demyelination of cerebellar slices.

<p><b>(A)</b> Bar graph illustrating changes in MBP, MOG and NFH staining after psychosine (100 nM) ± DEDA (100 nM) treatments. <b>(B)</b> Representative confocal images displaying MBP (MBP, green), MOG (MOG, yellow) and neurofilament (NFH, red) immunostaining under treatment conditions indicated. Confocal images captured at ×10 magnification; scale bar 200μm. Mean fluorescence was calculated using a total of 25–36 independent ROI observations in each experiment. Data are presented as mean±s.e.m. (n = 7), one-way ANOVA and Newman–Keuls multiple comparison post-test *p<0.05; #p<0.05 comparing control ± psychosine.</p

cPLA2 inhibitor BDDPAA, but not sPLA2 inhibitor Varespladib, attenuates psychosine-induced toxicity in human astrocytes.

<p>Human astrocytes were pre-treated with <b>(A)</b> the specific sPLA2 inhibitor Varespladib (10μM for 1h) (n = 4) or <b>(B)</b> the specific cPLA2 inhibitor BDDPAA (1μM for 1h) (n = 4–5) before treatment with psychosine (10μM for 3h or 20h). Cell viability was quantified by an MTT assay. Data is presented as mean + s.e.m. The main graph shows the ratio between control vs psychosine and drug vs drug+psychosine and smaller graphs show comparisons to vehicle control and psychosine treatment.</p

DEDA treatment attenuated the psychosine-induced axonal damage.

<p>Relative surface area of SMI-32 axonal expression <b>(B)</b> shows a significant increase in the white matter tracts induced by psychosine treatment, and attenuated by DEDA co-treatment. <b>(A)</b> Representative images of SMI-32 expression in cerebellar slice culture. Confocal images taken at 10x magnification; scale bar 200μm. Graphs expressed as mean±s.e.m. (n = 6), repeated measures one-way ANOVA, Newman-Keuls post-hoc test performed. ***p<0.0001 compared to control, ^###p< 0.0001 compared to psychosine treatment.</p

PLA2 inhibitor DEDA protects against psychosine-induced toxicity in human astrocytes.

<p><b>(A)</b> Experimental timeline depicting astrocytes were incubated in serum free media for 3h and treated with or without DEDA for 1h before addition of psychosine. Psychosine was used at concentrations indicated for 24h or with 10μM psychosine at incubation times indicated. <b>(B)</b> Psychosine induced a decrease in both mouse (<i>upper graph</i>) and human (<i>lower graph</i>) astrocytes cell viability in a concentration dependent manner (n = 5). <b>(C)</b> Human astrocytes were pre-treated with or without the PLA2 inhibitor DEDA (5μM for 1h) before treatment with 10μM psychosine at different incubation times (n = 4). <b>(D)</b> Human astrocytes were pre-treated with or without the PLA2 inhibitor DEDA (5μM for 1h) before treatment with different concentrations of psychosine for 3h (n = 3), 12h (n = 4) and 24h (n = 4). Cell viability was quantified by measuring NADP(H) activity using an MTT assay (Vybrant® MTT Cell Proliferation Assay Kit, Life technologies). Graphical data are presented as mean ± s.e.m. Unpaired Student’s t-test *p<0.05, **p<0.001 comparing psychosine ± DEDA.</p

DEDA inhibits psychosine-induced loss of vimentin expression in cerebellar slices.

<p><b>(A)</b> Organotypic slice cultures were prepared from the cerebellum of P10 mice and grown in culture for 12 days. Slices were treated with psychosine (100nM) and/or DEDA (100nM) for 18 h. The medium was then changed and DEDA treatment continued for a further 30 h, or control medium was added for this time. Cerebellar cultures were then processed for immunocytochemistry. <b>(B)</b> Bar graphs illustrate changes in vimentin and Iba1 staining after psychosine (100 nM) ± DEDA (100 nM) treatments. <b>(C)</b> Representative confocal images displaying the astrocyte marker vimentin (yellow) and <b>(D)</b> the microglia marker Iba1 (purple) immunostaining under treatment conditions indicated. Confocal images captured at ×10 magnification. Confocal images were taken at 10x magnification; scale bar 200μm. Mean fluorescence was calculated using a total of 25–36 independent ROI observations in each experiment. Data are presented as mean ± SEM. (n = 8), one-way ANOVA and Newman–Keuls multiple comparison post-test *p<0.05; #p<0.05 comparing control ± psychosine.</p

Psychosine-induced changes vimentin in human astrocytes and is partially reversed by DEDA.

<p>Human astrocytes were pre-treated with the PLA2 inhibitor DEDA (5μM for 1h) before treatment with psychosine (10μM for 3h or 24h). Representative confocal images displaying Hoesth (blue) and vimentin (red) staining under treatment conditions indicated. A 50μm concentric circle from the nucleus was drawn and the number of cellular extension beyond this were counted. A total of 20–30 cells were counted for each condition. Graphical data are presented as mean ± s.e.m. One-way ANOVA and Newman-Keuls Multiple Comparison post-test, compared to control (*p<0.05, **p<0.01, ***p<0.005) and compared to psychosine treatment (#p<0.05, ##p<0.01, ###p<0.005).</p

Extracellular, but not intracellular, calcium is required for psychosine-induced human astrocyte cell death.

<p>Human astrocytes were pre-treated with <b>(A)</b> the calcium chelator EDTA (1mM for 1h) (n = 4–6) or <b>(B)</b> the ryanodine receptor antagonist Dandrolene (10μM for 1h) (n = 4) before treatment with psychosine (10μM for 3h or 20h). Cell viability was quantified by an MTT assay. Data is presented as mean + s.e.m. The main graph shows the ratio between control vs psychosine and drug vs drug+psychosine and smaller graphs show comparisons to vehicle control and psychosine treatment.</p