Additional file 1 of Schwann cell-derived extracellular vesicles promote memory impairment associated with chronic neuropathic pain

Supplementary Material

Behavioral alteration in mice with chronic i.n. LPS administration.

<p>a) The ambulatory motility (VM=voluntary movement), Motor behavior was analyzed in an open-field test at the 1st, 3rd and 5th months after LPS instillation. b) Adhesive removal test, the asymmetry of left and right movement was detected by the adhesive removal test at the 5th month after LPS instillation. c) Latency to find platforms (from day 1 to day 5), the cognition was measured by Morris water maze at the 5th month after LPS instillation. Left=time to remove adhesive dots from left forelimb; Right= time to remove adhesive dots from right forelimb. Bars indicate SD for 7-10 mice at each point. *<i>P</i><0.05 compared with the contralateral side and/or saline control, respectively. </p

Pathological changes in mice subjected to chronic i.n. LPS administration.

<p>a) Loss of dopaminergic neurons in the SN of mice challenged with right intranasal LPS administration of 10 µg/ every other day for 5 months; b) DA neurons in the VTA region on both sides; c) Representative photomicrographs illustrating TH immunoreactivity in the striatum; d) Neurons in the hippocampus and cortex were immunostained with anti-NeuN antibodies. Bars indicate SD for 7-10 mice. **<i>P</i><0.01 compared with contralateral side and saline control, respectively; scale bar is 2 mm. </p

Western blot analyses of TH and ChAT in protein extracts from the left and right sides of the SN in mice challenged with right intranasal LPS (10 µg/every other day) or saline administration of for 5 months.

<p>Semi-quantification of relative TH and ChAT protein was obtained from among at least six different animals. *<i>P</i><0.05 and ***<i>P</i> <0.01 compared with the contralateral SN and saline control, respectively. </p

The influence of peripheral immune on mice with chronic i.n. LPS.

<p>At the 5th month after LPS inoculation, mice were sacrificed and splenic mononuclear cells and the serum were isolated. The viability of T cells, oxidation products and inflammatory cytokines from spleens were detected in the presence and/or absence of α-synuclein stimulation by MTT, Nitrite and ELISA assays, respectively. Inflammatory cytokines from the serum were detected by ELISA assays. a) The viability of T cells; b) NO production, c) The secretion of inflammatory cytokines IL-1β, IL-6 and TNF-α; d) the secretion of Th1 IFN-γ, Th2 IL-10 and Th17 IL-17 and e) The secretion of inflammatory cytokines IL-1β, IL-6, IL-10 and TNF-α from the serum of the mice. There was no statistical significance between and within two groups. Determinations were performed in duplicate and results were expressed as mean±S.E.M. from at least six mice.</p

Accumulation and aggregation of α-synuclein were determined by Western blot and immunostaining.

<p>a) Western blot, semi-quantification of α-synuclein protein was shown in histogram. b) Three-labeled immunofluorescence (Green=TH; Red=α-synuclein and Blue=Hochest) and quantitative analysis of α-synuclein⁺ cells within the rectangles; scale bar is 2 mm. c) Accumulation and aggregation of α-synuclein in the SN. The expression of insoluble α-synuclein (red) after PK treatment around the nucleus (blue); scale bar is 200 μm. Semi-quantification of α-synuclein protein was obtained from among at least six different animals. *<i>P</i><0.05 compared with the contralateral SN and saline control, respectively. </p

The changes of the striatal biogenic amines following chronic i.n.

<div><p><b>LPS in mice</b>. </p> <p>The levels of DA and its major metabolites DOPAC and HVA, as well as NE, Iso, 5-HT and 5-HIAA in SN were determined by specific HPLC assay at the 5th month after LPS inoculation. a) DA; b) DOPAC; c) HVA and d) the ration of HVA/DA; e) NE; f) Iso; g) 5-HT; h) 5-HIAA. Quantitative results were obtained from among at least six mice. *<i>P</i><0.05 and **<i>P</i><0.01 compared with the contralateral striatum and saline control, respectively.</p></div

Microglia activation in the SN in mice after chronic i.n. LPS exposure.

<p>a) Immunohistochemical evidence for F4/80 (green) and p-NF-<b>к</b>B/p65 (red) expression in the SN of mice challenged with right intranasal LPS administration of 10 µg/ every other day for 5 months; Scale bar is 500 µm; b) TNF-α, IL-1β and IL-6 expression in the SN determined by ELISA assays. ***<i>P</i><0.001, compared with the contralateral side and saline control, respectively; *<i>P</i><0.05, compared with the contralateral side and saline control. </p

Cell viability was tested with CCK-8

<p>. * P<0.05 vs. the N group; # P<0.05 vs. the H group; Δ P<0.05 vs. the H+PF20 group. Mean ± SD, n = 6.</p

Chemical structure of paeoniflorin.

<p>Chemical structure of paeoniflorin.</p