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

Loss of spiral ganglion neurons in mice infected in newborn period with MCMV.

<p>Mice were sacrificed at various postnatal days (PNd) 7, 11, and 30 as described in the Material and Methods. For each time point, cochlea were removed and placed in 4% paraformaldehyde, decalcified and embedded in paraffin as described in Materials and Methods. Sections were stained with anti-Tuji 1 antibodies to detect neurons followed by horseradish peroxidase (HRP) conjugated second antibody and development with diaminobenzidine (brown). Cresyl violet (CV) was used as counterstain. Digitalized images, of the inner ear from each individual tsection (2 per cochlea) were imported into the <i>B-Cell Image</i> program. Spiral ganglion cell (SGC) densities were calculated in infected animals (4 mice per time point) and mock infected animals (3 mice per time point) by determining the number of spiral ganglion neurons followed by normalization to the area of Rosenthal’s canal in each section to provide the spiral ganglion neuron density (cells/mm²). Spiral ganglion neuron densities in the basal, mid and apical turns (I, II, III) of the cochlea are presented as the mean number of neuron density with error bars demarcating SEM (<b>**</b> indicates significant differences p<0.0022, and *** p<0.001 by Mann-Whitney).</p

Detection of MCMV infected cells in the inner ear of MCMV infected mice.

<p>Infected cells in temporal bone marrow <b>(A)</b>, in spiral ganglion <b>(B,C)</b> and <b>(D-F)</b> in lateral wall of cochlea <b>(D-F)</b>. Panels A (x60), B,F (x40), C,D,E (x20) and boxed areas digitally zoomed in right lower corner. Panels D,E demonstrate infected cells in the spiral ligament and Panels E, F illustrate infected basal cells in the stria vascularis. Newborn mice infected with 200PFU of Smith strain MCMV were sacrificed on postnatal day (PNd) 11 and after exhaustive perfusion and fixation, the inner ears were removed, decalcified and embedded in paraffin as described in Materials and Methods. Sections were stained with anti-MCMV IE-1 (pp89) antibody CROMA 101 and visualized using a horseradish peroxidase (HRP) conjugated second antibody followed by development with diaminobenzidine (brown). Virus infected (IE-1+) cells were detected in inner ear sections from 5/8 animals examined. Scale bars indicating 50u (A), 100u (B, F,), and 200u (C,D,E) are shown at bottom left of figures.</p

Progressive hearing loss and virus inoculum effect on progression of hearing loss in MCMV infected mice.

<p><b>(A)</b> MCMV infected mice exhibit increased ABR threshold as function of age. Newborn mice infected with uncloned Smith MCMV(●; n = 12) or mock infected (<b>○;</b> n = 15) were tested at approximately 3,7, and 12 months and ABR thresholds determined. Note increasing mean ABR threshold in infected mice at 7 months of age and increase in ABR threshold for both infected and mock infected groups of mice at 12 months of age. At each time point the mean ABR thresholds of mock vs. infected mice were significantly different (p<0.001 two-way Anova with Bonferroni post-test correction). (<b>B)</b> Dose dependence of progressive hearing loss and delayed onset of hearing loss. Newborn mice were inoculated with recombinant, BAC cloned MCMV(50PFU ●; 200PFU ▲) or mock uninfected (<b>○</b>) and tested for auditory function at PNd42 or PNd78. Each symbol represents one ear tested (n = 96 mock, n = 36(50PFU), n = 52(200PFU)). Note the increase in median ABR threshold in mice given 50 PFU between PNd42 and PNd78 as compared to mock infected animals. (<b>**</b>) indicates significant differences (p<0.01) between groups with increase in ABR thresholds as determined by Kruskal-Wallis test of medians with Dunn post-test correction. <b>(C)</b> Magnitude of ABR thresholds in cochlea with progression of hearing loss. The change (dB) in individual cochlea exhibiting an increase in ABR threshold between d42 and d78 (Δ ABR threshold) was plotted for each group and medians compared. Number of cochlea with progression that were analyzed were (mock = 27; 50PFU = 13; 200PFU = 26). Statistically significant differences between median Δ ABR threshold of mock vs 50PFU, (p<0.01) and mock vs. 200PFU (p<0.001) by Kruskal-Wallis test of medians with Dunn post-test correction.</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

Persistent expression of proinflammatory chemokines in cochlea of mice with ABR thresholds >60dB.

<p>Mice were infected with MCMV or mock infected as controls as described in Materials and Methods. One group of infected mice (n = 12) and control mice (n = 6) were harvested at PNd10. A second group underwent ABR testing on PNd42 and equal numbers (n = 12) of mice with ABR thresholds >60dB (median 75dB) or <60dB (median 45dB) were harvested, cochlea pooled from individual mice and expression of proinflammatory chemokines determined as described in Materials and Methods. Results are reported as fold change and statistical comparison to age matched control mice (p value). Note increased expression of CCL8, CXCL9, and CXCL10 in mice with hearing loss.</p><p>Persistent expression of proinflammatory chemokines in cochlea of mice with ABR thresholds >60dB.</p

Detection of MCMV viral DNA in cochlea of mice infected as newborns with MCMV.

<p><b>(A)</b> Newborn mice were inoculated intraperitoneally with 200PFU of Smith strain of MCMV and at the indicated times, mice were sacrificed, exhaustively perfused and cochlea removed. Viral DNA was detected using a quantitative PCR as described in Materials and Methods and expressed as log genome copies per mg of cochlear tissue. The limit of detection of this PCR assay was defined through comparison of the signal derived from DNA extracted from the cochlea of age-matched mock infected mice and was found to be less than 10 copies per mg of extracted tissue. Age post infection is shown on x-axis. On PNd14, cochlea from individual mice were pooled (n = 5) whereas at PNd120 (n = 9 mice) and 12 months (n = 5 mice), each symbol represents single cochlea. Note that results from cochlea of infected mice on PNd120 and 12 months that were below limits of detection of this assay were plotted as log = 0. The signal detected in tissue from mock infected animals was below the level of detection. <b>(B)</b> Newborn mice were inoculated with different amounts of recombinant BAC cloned Smith strain MCMV as indicated. On PNd8, cochlea (n = 12) were harvested from mice as above and the quantity of MCMV DNA (log genome copy) per mg of tissue determined as described above. Horizontal bar depicts median of total group and (*) indicates statistically significant difference in signals (50PFU vs 100PFU, p = 0.016; 50PFU vs 200PFU, p = 0.0003; 100PFU vs 200PFU, NS p = 0.215; Kruskal-Wallis test of medians with Dunn post-test correction). No signal was detected from cochlear DNA extracted from mock infected animals (n = 12 cochlea).</p

Mononuclear cells infiltrates in the cochlea of infected mice.

<p><b>(A)</b> Mononuclear cell infiltrate in the cochlea of infected mice. H&E staining of cochlea demonstrates mononuclear cellular infiltrates in the stria vascularis (arrow). <b>(B,C)</b> Immunohistochemical detection of CD3+ mononuclear cells in spiral ganglion (B) and stria vascularis(C) (20x). Cochlea from infected mice were harvested a PNd11 and following extensive perfusion, cochlea were fixed and sections for immunohistochemistry as described in Materials and Methods. Staining with anti-CD3 antibodies, detection with a HRP conjugated secondary antibody and development with AEC was carried as described in Materials and Methods. Arrows depict CD3+ cells with digital zoom boxed in lower right corner an scale bars 200um in left corner. <b>(D)</b> Detection of CD3+ mononuclear cells in spiral ganglion of MCMV infected mouse with hearing loss. Frozen section from the cochlea from PNd120 mouse with hearing loss documented on PNd50 was reacted with rabbit anti-CD3 and mouse anti-Tuj1 antibodies, followed by staining with FITC anti-rabbit IgG secondary antibodies or tetramethylrhodamine isothiocyanate conjugated anti-mouse IgG secondary antibodies and viewed under confocal microscope (40x). Note CD3+ cells (green) and Tuj1 positive cells (red) neurons in boxed area (60x) in lower right hand corner.</p