FIGURES AND TABLES
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Induction of cytomegalovirus-infected labyrinthitis in newborn mice by lipopolysaccharide: a model for hearing loss in congenital CMV infection
Li Li, Isao Kosugi, Gui-Ping Han, Hideya Kawasaki, Yoshifumi Arai, Tamotsu Takeshita and Yoshihiro Tsutsui
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Figure 1.
Survival rate of newborn mice i.p. infected with MCMV in combination with i.c. injections of LPS or PBS. Newborn BALB/c mice were i.p. inoculated with MCMV (1 
105 PFU) 2 days after birth. At 1 day before the infection, LPS (5 
g/5 l) was injected into the bilateral brains. After MCMV infection, the same dose of LPS was injected into the other side of the brains alternatively three times or left without additional injection of LPS. Control mice were injected with PBS in the same way after i.p. infection with MCMV. Survival rates of mock-infected newborn mice that were injected one or four times with LPS are also shown.
Figure 2.
Viral titers of the inner ears and the brains in mice i.p. infected with MCMV and one or four times i.c. injections of LPS or PBS. The inner ear tissues and brains were excised 6 days after infection and weighed and stored at -
80°C. The tissues were homogenized in 500 l of MEM. A part of the supernatant from each sample was subjected to a plaque assay. Viral titers were expressed as PFU per milligram of organ. *
P<0.01 by Student's t-test. **
P<0.05 by Student's t-test.
Figure 3.
Localization of MCMV IE1-positive cells in the labyrinths in the MCMV-i.p.-infected mice with i.c. injections of LPS. Newborn mice were i.p. infected with MCMV and received i.c. injection of LPS four times. Mice were killed 6 days after infection, and serial sections of the inner ears were made after fixation and decalcification. Sections were reacted with anti-IE1 mAb, incubated with horseradish peroxidase-labeled secondary Ab, and colored with DAB. (a) Low-power view of the labyrinth, HE stain. CN, cochlear nerve; M, mesenchymal region; SG, spiral ganglia; ST, scala tympani; SV, scala vestibuli; SM, scala media; CO, Corti organ. (b) IE1-positive cells in the adjacent section. IE1-positive cells were observed in the following higher power views: (c) in the mesenchymal region, (d) along the cochlear nerve (arrows), (e) in the epithelial cells of ST, (f) in the spiral limbus (SL), (g) in the cerebral ventricular wall. VZ, ventricular zone; SVZ, subventricular zone and (h) in the cerebral meninges (Men).
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Figure 4.
Distribution of IE1-positive cells in the labyrinths in the MCMV-i.p.-infected mice with i.c. injections of LPS (a) and in the MCMV-i.c.-infected mice (b). In both groups, newborn mice were infected and killed 6 days after infection. After immunohistochemical staining, IE1-positive cells were counted in the portions of the labyrinths, such as cochlear nerve (CN), mesenchymal (M) region, including spiral ganglia (SG), scala tympani (ST), scala vestibuli (SV), spiral limbus (SL) and scala media (SM). The number of IE1-positive cells was expressed as the average number
s.d. per labyrinth. Virus-infected cells were counted in three sections per each animal and at least eight animals were assessed. *
P<0.05 by Student's t-test. #Only two cases had positive cells in this lesion.
Figure 5.
Localization of IE1-positive cells in the labyrinths in the MCMV-i.c.-infected mice. Newborn mice were i.c. infected with MCMV and killed 6 days after infection. (a) Low-power view of labyrinth, HE stain. SV, scala vestibuli; (b) IE1-positive cells in the adjacent section. Men, meninges; Po, pons. (c) IE1-positive cells in scala tympani (ST), and lack of infected cells in scala media (SM), including Corti organ (CO). (d) IE1-positive cells along the cochlear nerve (CN).
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Figure 6.
Immunohistochemical double staining of the labyrinths with antibodies against the viral antigen and antibodies specific to myeloperoxidase (MPO) (a), factor VIII (b), or neuronal nuclei (NeuN) (c) in the MCMV-i.p.-infected mice with LPS. (a) Sections were reacted with anti-IE1 mAb, incubated with secondary Ab, and colored with AEC, then reacted with anti-MPO pAb and incubated with ALP-conjugated secondary Ab, colored with Fast Blue. M, mesenchymal region; SV, scala vestibuli. (b) Sections were reacted with anti-IE1 mAb, incubated with secondary Ab, and colored with AEC, then reacted with anti-factor VIII pAb and incubated with ALP-conjugated secondary Ab and colored with Fast Blue. Arrows show double-stained cells. ST, scala tympani. (c) Sections were reacted with anti-Q3 (cytoplasmic antigen) mAb, incubated with secondary Ab, and colored with AEC, then sections were reacted with anti-NeuN mAb and incubated with ALP-conjugated secondary Ab and colored with Fast Blue. SG, spiral ganglia; SM, scala media.
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Figure 7.
Typical profiles of auditory brainstem response (ABR) of mock-infected and MCMV-i.p.-infected mice with i.c. injection of LPS 4 weeks after infection. In mock-infected mouse (a) ABR disappeared at 35 dB SPL, while in MCMV-i.p.-infected mouse with i.c. injections of LPS (b) ABR disappeared at 80 dB SPL and the amplitude of ABR was lower than that in control mouse at any SPL.
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