7 research outputs found
Infiltration of inflammatory cells and induction of proinflammatory cytokines in the brains of MCMV infected mice.
<p>A. Percentage of CD45<sup>hi/int</sup>, F4/80<sup>+</sup> mononuclear cells in the brain following infection with MCMV, PND8. Plots are representative of 1 of 4 replicates, n = 4 mice pooled/replicate. B. Expression of MHC Class II, gated on CD45<sup>hi/int</sup>, F4/80<sup>+</sup> population. Histogram is representative of 1 of 4 replicates, n = 4 mice pooled/replicate. C. Expression of Iba-1 (red), a marker for activated macrophages/microglia, and TOPROIII (blue), a nuclear marker, in the cerebellum of control and infected mice at PND8, 20×, scale bars = 50 µm. The number of Iba-1<sup>+</sup> cells was quantified from 4 sections/animals, n = 8 mice/experimental group. Data are shown as mean +/− SEM. P values were calculated using a two-tailed T test. D. Inflammatory gene expression in the cerebellum of control and infected mice at PND8. Data are shown as mean +/− SEM. P values were calculated using a two-tailed T test, n = 5 mice/experimental group.</p
Treatment with the glucocorticoid prednisolone normalizes granule neuron progenitor cell proliferation in MCMV infected mice.
<p>A. Representative images of brain sections depicting the expression of cell cycle markers in the EGL of control or infected mice treated with vehicle or pred; BrdU (green), Ki67 (red), TOPROIII (blue), 60×, scale bars = 20 µm. B–C. Stereological quantification of BrdU<sup>+</sup> and Ki67<sup>+</sup> GNPCs in the EGL of vehicle or pred treated, control and infected mice. Data are shown as mean +/− SEM, 8 sections were counted per mouse, n = 5–6 mice/experimental group. P values calculated using two-way ANOVA. Vehicle treated control vs. MCMV were significantly different (p≤.001) as determined by two-tailed T test. D. (Top) Detection of phospho-cyclin B1 and cyclin B1 in the cerebellum by immunoblotting. Actin loading control shown at bottom. Each lane represents 2 cerebella pooled, n = 2 lanes/experimental group. (Bottom) Densitometry showing the expression of p-cyclin B1, relative to actin, in the cerebellum of vehicle or pred treated, control and infected animals. Data are representative of 3 replicate blots. P value calculated by two-way ANOVA. Control vs. MCMV were significantly different (p≤.02) as determine by two-tailed T test.</p
Treatment with the glucocorticoid dexamethasone normalizes developmental gene expression in the brains of infected mice but leads to cerebellar hypoplasia.
<p>A. Cerebellar expression of developmentally regulated genes from uninfected and infected mice treated with vehicle or dexa analyzed by quantitative real-time PCR. Data are shown as mean +/− SEM, fold change normalized to control = 1, n = 5 mice/experimental group. P values calculated using two-way ANOVA. B. Quantification of cerebellar area (expressed as a percentage of total brain area) in mice treated with vehicle or dexa. Data are shown as mean +/− SEM, measurements were taken from 5 sections/mouse, n = 5–7 mice/experimental group. P values were calculated by two-way ANOVA.</p
Neonatal infection with MCMV results in a focal encephalitis with global deficits within the cerebellum.
<p>A. Expression of immediate early gene 1, protein pp89 (IE-1) (green) in the cerebellum, a non-structural protein encoded by MCMV very early in infection, PND8, 60×. Note the focal nature of infection in the external granule cell layer (EGL) of the cerebellar cortex. B. Cresyl violet staining showing a global effect of virus infection on cerebellar area and folia development, 4×. Note the smaller size, delayed foliation and delayed fissure formation in the cerebellum of infected animals.</p
Treatment with the glucocorticoid prednisolone limits altered morphogenesis and developmental gene expression in the cerebella of MCMV infected mice.
<p>A. Cerebellar area of control or MCMV infected mice treated with vehicle or pred. Data are shown as mean +/− SEM. Stereological measurements from 5 sections/mouse, n = 5 mice/experimental group. P values calculated using two-way ANOVA. B. External granule cell layer (EGL) thickness in vehicle or pred treated control and MCMV infected mice. Data are shown as mean +/− SEM. EGL thickness was determined from 4 measurements/section, 8 sections/mouse, n = 6–8 mice/experimental group. P values calculated using two-way ANOVA. C. Granule neuron progenitor cells (GNPCs) in the cerebella of control and infected mice treated with vehicle or pred. Data are shown as mean +/− SEM. GNPC numbers from 8 sections were counted per mouse, n = 5–6 mice/experimental group. P values calculated using two-way ANOVA. D. Representative cerebellar sections showing a thickening of the EGL following MCMV infection that is corrected with pred treatment, 60×, scale bars = 20 µm. EGL containing GNPCs are shown in white, the adjacent molecular layer is shown in black. E. Transcription of developmentally regulated genes within the cerebellum of vehicle or pred treated, control and infected mice. Data are shown as mean +/− SEM. Fold change normalized to control = 1, n = 5 mice/experimental group. P values calculated using two-way ANOVA.</p
Treatment with prednisolone decreases the infiltration of inflammatory cells into the brain of MCMV infected mice.
<p>A. Schematic showing the time course of MCMV infection and prednisolone (pred) treatment. B. Quantitative real-time PCR for IE-1 showing viral genome copy number in the liver, spleen, brain and cerebellum of infected mice treated with vehicle or pred. P values calculated by Mann-Whitney test, n = 22–25 mice/experimental group for liver, spleen and brain; n = 5–7 mice/experimental group for cerebellum. C. (Top) Quantification of Iba-1+ cells in the cerebellum of vehicle or pred treated, control and infected mice. Data shown as mean +/− SEM. P values calculated by two-way ANOVA. Iba-1<sup>+</sup> cells were counted in 4 sections/animal, n = 6 mice/experimental group. (Bottom) Panels of representative sections analyzed by confocal microscopy showing Iba-1 staining (red) and TOPROIII (blue) in the cerebellum of control or infected mice treated with vehicle or pred. Panel below represents black and white rendering of immunofluorescent images to increase contrast, white signals represent Iba-1<sup>+</sup> cells. All images taken at 20×, scale bars = 50 µm. D (Left) Representative dot plots showing the percentage of CD45<sup>lo</sup> and CD45<sup>hi/int</sup>, F4/80<sup>+</sup> macrophages in the brain of vehicle or pred treated animals, gated on mononuclear cells. Plots are representative of 1 of at least 3 replicates, n = 4 mice pooled/replicate. (Right) Bar graphs showing the percentage of mononuclear cells that are CD45<sup>lo</sup> and CD45<sup>hi/int</sup>, F4/80<sup>+</sup> macrophages in the CNS. Data are shown as mean +/− SEM, n = 4 mice pooled/replicate, 3–5 replicates/experimental group. P values were calculated using two-way ANOVA.</p
Treatment of MCMV infected neonates with dexamethasone decreases infiltration of inflammatory cells and expression of interferon stimulated genes in the CNS without increasing levels of virus replication.
<p>A. Infectivity assay showing viral titers in the liver, spleen and brain of infected mice treated with vehicle or dexamethasone (dexa). Each circle represents plaque forming units (PFU)/mg of tissue for an individual animal, p values calculated using two-tailed T test. B. (Top) Dot plots showing the percentage of CD45<sup>lo</sup> and CD45<sup>hi/int</sup>, F4/80<sup>+</sup> mononuclear cells in the brain of infected animals following treatment with dexa, gated on mononuclear cells. Plots are representative of 1 of 4 replicates. (Bottom) Bar graphs showing the percent of CNS mononuclear cells that are CD45<sup>lo</sup> and CD45<sup>hi/int</sup>, F4/80+ macrophages. Data are shown as mean +/− SEM, n = 4 mice pooled/replicate, 4 replicates/experimental group. P values calculated using one-way ANOVA. C. (Top) Bar graph depicting the number of Iba-1<sup>+</sup> cells within the cerebellum of vehicle treated or dexa treated MCMV infected mice. Data are shown as mean +/− SEM. The number of Iba-1<sup>+</sup> cells was quantified from 4 sections/animal, n = 5–8 mice/experimental group. P values calculated using two-way ANOVA. (Bottom) Representative Iba-1 staining depicting activated macrophages within the cerebellum of vehicle treated or dexa treated infected mice, PND8, 20× scale bars = 50 µm. (Left) Iba-1-red, TOPROIII-blue, (Right) Black and white rendering of immunofluorescent images to increase contrast, white signals represent Iba-1 staining. D. Quantitative real-time PCR analysis of transcription of IFIT1, IFIT2 and STAT1 in the cerebellum of infected mice following treatment with dexa. Data are shown as mean +/− SEM, fold change normalized to control = 1, n = 5 mice/experimental group. P values calculated using two-way ANOVA.</p