DataSheet_1_Characterization of Pathogenesis and Inflammatory Responses to Experimental Parechovirus Encephalitis.pdf

Human parechovirus type 3 (PeV-A3) infection has been recognized as an emerging etiologic factor causing severe nerve disease or sepsis in infants and young children. But the neuropathogenic mechanisms of PeV-A3 remain unknown. To understand the pathogenesis of PeV-A3 infection in the neuronal system, PeV-A3-mediated cytopathic effects were analyzed in human glioblastoma cells and neuroblastoma cells. PeV-A3 induced interferons and inflammatory cytokine expression in these neuronal cells. The pronounced cytopathic effects accompanied with activation of death signaling pathways of apoptosis, autophagy, and pyroptosis were detected. A new experimental disease model of parechovirus encephalitis was established. In the disease model, intracranial inoculation with PeV-A3 in C57BL/6 neonatal mice showed body weight loss, hindlimb paralysis, and approximately 20% mortality. PeV-A3 infection in the hippocampus and cortex regions of the neonatal mouse brain was revealed. Mechanistic assay supported the in vitro results, indicating detection of PeV-A3 replication, inflammatory cytokine expression, and death signaling transduction in mouse brain tissues. These in vitro and in vivo studies revealed that the activation of death signaling and inflammation responses is involved in PeV-A3-mediated neurological disorders. The present results might account for some of the PeV-A3-associated clinical manifestations.</p

Average body weight at different ages.

<p>The pups received either normal saline (NS) or dexamethasone (Dex) at P7. Body weight was recorded before drug administration (P7), at weaning (P21), and at the time of the behavioral tests (P56). The rats continued to gain body weight with age, and there was no significant difference between NS and Dex groups. The data are expressed as mean ± SEM of three independent experiments (<i>n</i> = 5 for each group).</p

The effects of dexamethasone (Dex) are mediated through the PI3K and NF-κB pathway.

<p>Rat pups were intraperitoneally injected with Dex or normal saline (NS) at P7 and sacrificed at P56 immediately after behavioral tests. The cytosolic levels of PI3Kp110 (A), PI3Kp85 (B), and NF-κB (C) were significantly higher in the Dex group (PI3Kp110: 2.57 ± 0.22; PI3Kp85: 2.57 ± 0.19; NF-κB: 1.93 ± 0.13) than in the NS group (PI3Kp110: 0.90 ± 0.05; PI3Kp85: 0.85 ± 0.04; NF-κB: 0.90 ± 0.04) (Mann-Whitney test, *<i>p</i> < 0.05, **<i>p</i> < 0.01). The band intensities were normalized to tubulin. Results represent the mean ± SEM of three independent experiments (<i>n =</i> 10 for each group).</p

Effects of postnatal administration of dexamethasone (Dex) on fear memory and spatial learning memory formation.

<p>Dex was administered at P1 or P7 and behavioral tasks were performed at P56. (A) The freezing response in contextual fear-conditioning task was significantly greater in the Dex-P7 group than in the other groups (two-way ANOVA with Bonferroni multiple comparisons test, <i>n</i> = 8 for each group *<i>p</i> < 0.05), and Dex-P7 group was significantly greater than NS-P7 group (F[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165752#pone.0165752.ref001" target="_blank">1</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165752#pone.0165752.ref028" target="_blank">28</a>] = 4.59, *<i>p</i> < 0.05). (B) The freezing response in the cued fear-conditioning task was not significantly altered among the four groups (<i>n</i> = 8 for each group). (C) The proportion of time spent searching in target quadrant was significantly higher in the Dex group (64.50 ± 1.50) than in the NS group (48.75 ± 1.56) (Mann-Whitney test, <i>n</i> = 5 for each group, *<i>p</i> < 0.05). (D) The number of crossings in the platform location was higher in the Dex group (12.20 ± 0.22) than in the NS group (6.75 ± 0.55) (Mann-Whitney test, <i>n</i> = 5 for each group, *<i>p</i> < 0.05). The data are expressed as mean ± SEM of three independent experiments.</p

A single postnatal dose of dexamethasone (Dex) increases neurogenesis in the dentate gyrus.

<p>The rat pups were treated with NS or Dex at P7, followed by a single injection of BrdU (50 mg/kg, ip.) and sacrificed at P56. (A) To identify the neurogenesis in the dentate gyrus, double immunofluorescence staining was performed to detect NeuN (red color) and BrdU (green color). The dotted lines show the shape of dentate gyrus. Bar = 200 μm. (B) Quantification of neuronal differentiation of newly born cells. The percentage of BrdU-positive cells co-expressing NeuN was calculated, and the percentage of neuronal differentiation was significantly higher in the Dex group than in the NS group (Mann-Whitney test, *<i>p</i> < 0.05). Results are representative of the mean ± SEM (<i>n =</i> 5 for each group). (C) Quantification of co-localized cells in the dentate gyrus. Net neurogenesis was significantly higher in the Dex group than in the NS group (Mann-Whitney test, *<i>p</i> < 0.05). Results are representative of the mean ± SEM (<i>n =</i> 5 for each group).</p

The effect of dexamethasone (Dex) on synaptic plasticity in the hippocampus.

<p>The pups received either normal saline (NS) or Dex at P1 or P7, and were sacrificed at P56 after the behavioral tests. (A) The brain sections were Golgi-stained and photomicrographs show the dendrites of granular cells. (B) The dendritic spine density of granular cells was significantly higher in the Dex-P7 group than in the other groups (two-way ANOVA with Bonferroni multiple comparisons test, ***<i>p</i> < 0.001). (C, D) The total dendritic length (C) and total branch numbers of the dendrites (D) did not differ significantly among the four groups. The data are expressed as mean ± SEM of three independent experiments (<i>n</i> = 6 for each group). Bar = 10 μm.</p

A Single Postnatal Dose of Dexamethasone Enhances Memory of Rat Pups Later in Life - Fig 4

<p>(A) Western blot analysis shows the membranous levels of PSD-95 in the hippocampus of young-adult rats receiving dexamethasone (Dex) at P7. PSD-95 levels were significantly higher in the Dex group (1.90 ± 0.11) than in the NS group (1.00 ± 0.07) (Mann-Whitney test, *<i>p</i> < 0.05). Tubulin was used as an internal control. The data are expressed as mean ± SEM of three independent experiments (<i>n</i> = 6 for each group). Administration of dexamethasone (Dex) at P7 does not affect the membranous level of AMPA receptors in the hippocampus measured at P56. (B-E) The membranous level of AMPA receptor subunits, GluR1 (B) and GluR2 (C), did not differ significantly between the Dex and NS groups. The expression levels of NR2A (D) and NR2B (E) were significantly higher in the Dex group (NR2A: 1.97 ± 0.20; NR2B: 2.21 ± 0.24) than in the NS group (NR2A: 1.00 ± 0.05; NR2B: 1.00 ± 0.08) (Mann-Whitney test, *<i>p</i> < 0.05). Tubulin was used as an internal control. These measurements were done after the contextual or cue-feared conditioning. The data are expressed as mean ± SEM of three independent experiments (<i>n =</i> 6 each group).</p

A single postnatal dose of dexamethasone (Dex) increases cell survival in the dentate gyrus.

<p>The rat pups were treated with NS or Dex at P7 followed by a single injection of BrdU (50 mg/kg, ip.) and sacrificed at P56. Coronal sections were obtained and BrdU and identified with an anti-BrdU antibody. (A) Representative images show the dentate gyrus, and BrdU-positive nuclei are characterized as cell survival (green color). The dotted lines show the shape of dentate gyrus. Bar = 500 μm. (B) BrdU-positive nuclei were counted and the number of BrdU-positive nuclei was significantly higher in the Dex group than in the NS group (Mann-Whitney test, *<i>p</i> < 0.05). Results are representative of the mean ± SEM (<i>n =</i> 5 for each group).</p

The effect of dexamethasone (Dex) on cell proliferation of dentate gyrus at P7.

<p>The rat pups were intraperitoneally injected with either NS or Dex, followed by BrdU at P7 and sacrificed 24 h later. Representative images of BrdU-labeled cells (green color) are shown in (A). The dotted lines show the shape of dentate gyrus. Bar = 200 μm. (B) Quantification of BrdU-positive nuclei indicated greater cell proliferation in the Dex-treated group than in the NS group (Mann-Whitney test, *<i>p</i> < 0.05). Results are representative of the mean ± SEM (<i>n =</i> 5 for each group).</p

Structural and Dynamic Histomorphometric Parameters of Experimental Chronic Kidney Disease Rats that did or did not Undergo Parathyroidectomy.^*

<p>*N = 26 rats. Data reported as mean ± SD. Abbreviations: CKD, chronic kidney disease; PTX+CKD, parathyroidectomy and chronic kidney disease; MS/BS, percent mineralized bone surface; MAR, mineral apposition rate; BFR, bone formation rate; OV/BV, osteoid volume ratio; OS/BS, osteoid surface ratio; O.Th, osteoid thickness; OMT, osteoid maturation time</p><p>^ap < 0.01, compared with sham-operated control group.</p><p>^bp < 0.01, compared with CKD group.</p><p>^cp < 0.05, compared with sham-operated control group.</p><p>^dp < 0.05, compared with CKD group. NS, no significant (p > 0.05)</p><p>Structural and Dynamic Histomorphometric Parameters of Experimental Chronic Kidney Disease Rats that did or did not Undergo Parathyroidectomy.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133278#t001fn001" target="_blank">*</a>}</p