Presentation_1_Noise-induced hearing loss vulnerability in type III intermediate filament peripherin gene knockout mice.pdf

In the post-natal mouse cochlea, type II spiral ganglion neurons (SGNs) innervating the electromotile outer hair cells (OHCs) of the ‘cochlear amplifier' selectively express the type III intermediate filament peripherin gene (Prph). Immunolabeling showed that Prph knockout (KO) mice exhibited disruption of this (outer spiral bundle) afferent innervation, while the radial fiber (type I SGN) innervation of the inner hair cells (~95% of the SGN population) was retained. Functionality of the medial olivocochlear (MOC) efferent innervation of the OHCs was confirmed in the PrphKO, based on suppression of distortion product otoacoustic emissions (DPOAEs) via direct electrical stimulation. However, “contralateral suppression” of the MOC reflex neural circuit, evident as a rapid reduction in cubic DPOAE when noise is presented to the opposite ear in wildtype mice, was substantially disrupted in the PrphKO. Auditory brainstem response (ABR) measurements demonstrated that hearing sensitivity (thresholds and growth-functions) were indistinguishable between wildtype and PrphKO mice. Despite this comparability in sound transduction and strength of the afferent signal to the central auditory pathways, high-intensity, broadband noise exposure (108 dB SPL, 1 h) produced permanent high frequency hearing loss (24–32 kHz) in PrphKO mice but not the wildtype mice, consistent with the attenuated contralateral suppression of the PrphKO. These data support the postulate that auditory neurons expressing Prph contribute to the sensory arm of the otoprotective MOC feedback circuit.</p

Chemotherapy-induced astrocyte activation in the spinal cord dorsal horn.

<p>GFAP immunohistochemistry in L3-L5 spinal cord was carried out on day 13 post 1^st injection of paclitaxel (PTX), oxaliplatin (OXA) or saline (control). Column graph showing the percentage of GFAP immunoreactivity in spinal cord dorsal and ventral horns, and representative images depicting GFAP immunoreactivity in the dorsal horn. GFAP immunoreactivity was significantly higher in the dorsal horn of PTX-treated mice compared with saline controls (**P<0.01), with no significant changes in the ventral horn. Scale bar = 50μm. n = 4, one-way ANOVA followed by Bonferroni's multiple comparison’s test. Data are expressed as mean±SEM.</p

Changes in regulatory T-cell population in inguinal lymph nodes following chemotherapy.

<p>Flow cytometry of lymphocytes to characterise regulatory T-(T-reg)-cell changes in inguinal lymph nodes on days 7 and 13 post-1^st injection of paclitaxel (PTX), oxaliplatin (OXA) or saline (control) was carried out. CD4+ cells were first gated from lymphocyte singlets (A), followed by consecutive gating of CD25+FoxP3+ cells (B). Column graphs of CD4+CD25+FoxP3+ T-reg cell populations in the lymph nodes on day 7 (C) and day 13 (D) are expressed as a percentage of lymphocyte singlets. A significant decrease in T-reg cells was seen in OXA-treated mice on both day 7 (n = 6, ****P<0.0001) and day 13 (n = 4, *P<0.05) compared to saline control mice. One-way ANOVA followed by Bonferroni's multiple comparison’s test. Data are expressed as mean±SEM.</p

Systemic inflammatory changes in the blood and spleen.

<p>Flow cytometry of lymphocytes to characterise inflammatory changes in the blood and spleen was carried out on day 7 post-1^st paclitaxel (PTX), oxaliplatin (OXA) or saline (control) injection. Mononuclear cells were first gated (A), followed by consecutive gating of singlets (B). For blood, singlets were further gated for CD45+ lymphocytes (C). Representative flow cytometric plots and column graphs of CD45+CD4+ (D) and CD45+CD8+ (E) T-cell populations in the blood (expressed as a percentage of CD45+ lymphocyte singlets), and total IL-4+ lymphocytes (F) in the spleen (expressed as a percentage of lymphocyte singlets). Significant increase in CD45+CD4+ and CD45+CD8+ T-cell populations in the blood of PTX- and OXA- treated mice (n = 10), and IL-4 positive lymphocytes in the spleens of OXA-treated mice (n = 7–9) compared to saline controls were found. *P<0.05; **P<0.01; ***P<0.001 indicate significant difference between PTX/OXA and saline controls. One-way ANOVA followed by Bonferroni's multiple comparison’s test. Data are expressed as mean±SEM.</p

Chemotherapy-induced changes in microglia in the spinal cord.

<p>IBA-1 and P2ry12 immunohistochemistry in L3-L5 spinal cord segment was carried out on day 13 post-1^st injection of paclitaxel (PTX), oxaliplatin (OXA) or saline (control). (A) Column graph showing the percentage of IBA-1 immunoreactivity in the spinal cord dorsal and ventral horns, and representative images depicting IBA-1 immunoreactivity in the dorsal horn. No measurable changes in PTX (n = 4) or OXA (n = 3) group compared with saline (n = 4) were detected. Scale bar = 50 μm. (B) Column graph and representative images showing P2ry12 immunoreactivity in spinal cord dorsal and ventral horns, which was significantly lower in the PTX (n = 4) and OXA (n = 3) group, compared with saline controls (n = 3). Scale bar = 50 μm. ****P<0.0001 represents significant difference between PTX/OXA and saline in the dorsal horn. ^xxxP<0.001, ^xxxxP<0.0001 represent significant difference between PTX/OXA and saline in the ventral horn. One-way ANOVA followed by Bonferroni's multiple comparison’s test. Data are expressed as mean±SEM.</p

Effect of chemotherapy on mechanical sensitivity in C56BL/6J male mice.

<p>Time course of mechanical paw withdrawal threshold (PWT; in grams) of male C56BL/6J mice treated with paclitaxel (PTX), oxaliplatin (OXA) or saline (control). Measurements were taken from baseline before chemotherapy until day 27, with chemotherapy injections administered at days 0, 2, 4 and 6. Compared to baseline thresholds, significant decrease in PWT was seen on days 8, 13, and 16 in the PTX-treated group, and on days 8, 13, 16 and 27 in the OXA-treated group. **(P<0.01); ***(P<0.001); ****(P<0.0001) indicate significant difference between baseline and post-chemotherapy time points in the PTX group. ⁺⁺(P<0.01); ⁺⁺⁺(P<0.001); ⁺⁺⁺⁺(P<0.0001) indicate significant difference between baseline and post-chemotherapy time points in the OXA group. PTX (n = 16), OXA (n = 16), Saline (n = 15). RM two-way ANOVA followed by Bonferroni’s multiple comparisons test. Data are expressed as mean±SEM.</p

Chemotherapy-induced neuronal injury in the lumbar DRG.

<p>ATF-3, IB4, CGRP and NF200 immunohistochemistry in L3-L4 DRG was carried out on day 13 post-1^st injection of paclitaxel (PTX), oxaliplatin (OXA) or saline (control). (A) Column graph and representative images depicting neuronal ATF-3 expression in the DRG, which was significantly higher in the PTX group compared to saline control group (*P<0.05). Scale bar = 50 μm. (B) Column graph and representative images showing double immunolabelling of ATF-3 with IB4, CGRP and NF200 in the DRG of PTX-treated mice. ATF-3 was predominantly expressed in IB4+ and NF200+ DRG neurons in the PTX group (*P<0.05). Scale bar = 50μm. n = 3–4, one-way ANOVA followed by Bonferroni's multiple comparison’s test. Data are expressed as mean±SEM.</p

Cytokine and chemokine changes in the DRGs following chemotherapy.

<p>Cytokine and chemokine changes in the DRGs following chemotherapy.</p

Cytokine and chemokine changes in the spinal cord following chemotherapy.

<p>Cytokine and chemokine changes in the spinal cord following chemotherapy.</p