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^lo and CD45^hi/int, F4/80⁺ 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^lo and CD45^hi/int, 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⁺ cells within the cerebellum of vehicle treated or dexa treated MCMV infected mice. Data are shown as mean +/− SEM. The number of Iba-1⁺ 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

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⁺ 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⁺ cells. All images taken at 20×, scale bars = 50 µm. D (Left) Representative dot plots showing the percentage of CD45^lo and CD45^hi/int, F4/80⁺ 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^lo and CD45^hi/int, F4/80⁺ 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

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

Murine CMV-Induced Hearing Loss Is Associated with Inner Ear Inflammation and Loss of Spiral Ganglia Neurons

<div><p>Congenital human cytomegalovirus (HCMV) occurs in 0.5–1% of live births and approximately 10% of infected infants develop hearing loss. The mechanism(s) of hearing loss remain unknown. We developed a murine model of CMV induced hearing loss in which murine cytomegalovirus (MCMV) infection of newborn mice leads to hematogenous spread of virus to the inner ear, induction of inflammatory responses, and hearing loss. Characteristics of the hearing loss described in infants with congenital HCMV infection were observed including, delayed onset, progressive hearing loss, and unilateral hearing loss in this model and, these characteristics were viral inoculum dependent. Viral antigens were present in the inner ear as were CD3+ mononuclear cells in the spiral ganglion and stria vascularis. Spiral ganglion neuron density was decreased after infection, thus providing a mechanism for hearing loss. The lack of significant inner ear histopathology and persistence of inflammation in cochlea of mice with hearing loss raised the possibility that inflammation was a major component of the mechanism(s) of hearing loss in MCMV infected mice.</p></div

Infiltration of inflammatory cells and induction of proinflammatory cytokines in the brains of MCMV infected mice.

<p>A. Percentage of CD45^hi/int, F4/80⁺ 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^hi/int, F4/80⁺ 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⁺ 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 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

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

Virus inoculum dependence of ABR thresholds in individual ears of infected animals.

<p><b>(A)</b> Medians of ABR thresholds in best and worst ears of individual mice from mock infected and mice given 50PFU, 100PFU, and 200PFU (n = number of mice analyzed). Significant differences in best ear ABR threshold between mock infected and mice infected with 50PFU and 100PFU versus mice given 200PFU and worst ears of mice infected with 200PFU versus other groups (P<0.01 by Kruskal-Wallis test of medians with Dunn post-test correction). (B) Difference (ΔABR threshold) in ABR threshold between best and worst ear. Medians of differences in ABR thresholds for best and worst plotted as function of virus inoculum. Note that 24% and 30% of mice inoculated with 100PFU and 200PFU respectively have >15dB ABR threshold difference between best and worst ear as compared to 13% and 16% for mock infected mice and mice infected with 50PFU.</p

Levels of expression of proinflammatory chemokines in cochlea of infected mice are dependent on viral inoculum.

<p>Cochlear tissue isolated from infected and uninfected, control mice on PNd8 with number of mice in each indicated. Expression of individual genes determined as described in Materials and Methods and reported as fold change and statistical comparison with control group (p value). Note increasing fold change with increasing viral inoculum.</p><p>Levels of expression of proinflammatory chemokines in cochlea of infected mice are dependent on viral inoculum.</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⁺ and Ki67⁺ 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