Role of Adenosine A2A Receptor Signalling in Noise-Induced Cochlear Inflammation

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AIMS & HYPOTHESES

Aims

The principal aim of this thesis was to improve our understanding of the underlying cellular and molecular mechanisms and dynamics of the noise-induced inflammatory response in the cochlea, and to explore the role of adenosine A2A receptor signalling in noise-induced cochlear inflammation. The thesis was divided into three major studies, with the specific aims listed below.

Cochlear Inflammatory Response Associated with Acute Noise Exposure

This study aimed to characterise the inflammatory response in the mouse cochlea following acute exposure to traumatic noise. The specific aims were to: Examine the activation of the transcription factor nuclear factor-kappa B (NF-κB) in the noise-exposed cochlea.Determine the changes in gene expression levels of proinflammatory cytokines (TNF-α and IL-1β), chemokines (CCL2) and cell adhesion molecules (ICAM-1) in the cochlea following acute noise exposure.Characterise the distribution of the cell adhesion molecules, ICAM-1 and PECAM-1, in the cochlea, and the time course of their protein expression following acute noise exposure.

Cochlear Inflammatory Response Associated with Chronic Noise Exposure

This study aimed to characterise the inflammatory response in the mouse cochlea following chronic exposure to moderate noise. The specific aims were to:Determine the changes in gene expression levels of proinflammatory cytokines (TNF-α and IL-1β), chemokines (CCL2) and cell adhesion molecules (ICAM-1) in the cochlea following chronic noise exposure.Characterise the time course of ICAM-1 and PECAM-1 expression in the cochlea following chronic noise exposure.

Role of Adenosine A2A Receptor Signalling in Noise-Induced Cochlear Inflammation

This study aimed to ascertain the role of adenosine A2A receptors in the regulation of noiseinduced cochlear inflammation. The specific aims were to: Examine the expression and distribution of adenosine A2A receptors, including A2AR+ infiltrating cells, in the noise-exposed cochlea. Assess the cochlear inflammatory response in adenosine A2A receptor knockout (A2ARKO-/-) mice exposed to noise to determine the effect of adenosine A2A receptor gene deletion in noise-induced cochlear inflammation. Evaluate the anti-inflammatory effect of adenosine A2A receptor activation using the selective adenosine A2A receptor agonist, regadenoson.

Hypotheses

Acute exposure to traumatic noise induces an inflammatory response in the cochlea which includes excessive production of proinflammatory cytokines, chemokines, and cell adhesion molecules via the activation of the transcription factor NF-κB, followed by the recruitment of inflammatory cells and their infiltration in cochlear tissues.Chronic exposure to non-traumatic noise also induces an inflammatory response in the cochlea with increased expression of proinflammatory cytokines, chemokines, and cell adhesion molecules.Noise exposure increases the expression of adenosine A2A receptors in the cochlea as an endogenous protective mechanism (adaptive response) to limit inflammation.Deletion of the adenosine A2A receptor gene (A2ARKO-/- mice) leads to an enhanced inflammatory response in the noise-exposed cochlea.Post-exposure treatment with the selective adenosine A2A receptor agonist, regadenoson, suppresses inflammatory processes in the noise-exposed cochlea.

METHODS

Cochlear Inflammatory Response Associated with Acute Noise Exposure

The primary purpose of this initial study was to further our understanding of molecular mechanisms of the inflammatory response and to characterise the dynamics of the response in the C57BL/6 mouse cochlea in response to acute exposure to traumatic noise (acoustic trauma). This study also provided baseline measurements of inflammatory markers in the noise-exposed cochlea as a foundation for subsequent studies. Male C57BL/6 inbred mice (6 to 8 weeks) were exposed for 24 h to an open-field octave band noise (8-16 kHz) presented at a sound pressure level (SPL) of 100 decibels (dB). This is a traumatic noise level for mice that causes permanent hearing loss and is routinely used in our laboratory. To verify that these noise conditions produced permanent hearing loss, functional assessment of hearing acuity was performed prior to noise exposure (baseline measurements) and one month after noise exposure using auditory brainstem responses (ABRs) to acoustic clicks and tone pips (n = 4). Animals serving as controls (n = 8) for the study were exposed to ambient noise levels (55-65 dB SPL) in the animal holding facility. Initially, the early onset phase of the noise-induced cochlear inflammatory response was studied by examining the cochlear activation of the nuclear transcription factor, nuclear factor-kappa B (NF-κB). NF-κB is activated in response to diverse stimuli such as inflammation, infections, and other stressful situations, and regulates the expression of a number of proinflammatory mediators. Specifically, the nuclear activation of the two principal subunits, p65 and p50, which normally exist together as heterodimers in the cytoplasm of quiescent (unstimulated) cells, were examined. Nuclear translocation and activation of p65 and p50 in the cochlea was examined 6 h following noise exposure using immunofluorescence.As the next step, inflammatory influence of NF-κB signalling on the noise-induced damaged mouse cochlea was investigated by evaluating the expression of several key target genes of NF-κB, including proinflammatory cytokines (TNF-α and IL-1β), chemokines (CCL2) and cell adhesion molecules (ICAM-1 and PECAM-1). To study the time course (dynamics) of the inflammatory events in the cochlea, outcomes were assessed at various time intervals after the cessation of noise exposure: 6 h, 1 day (24 h), 3 days (72 h), and 7 days (168 h). All animals were randomly assigned to the experimental groups (post-exposure survival times), with each group consisting of eight animals (n = 8). This group size was based on our previous experience with functional and molecular studies in the cochlea. This number provides statistical power to obtain an 80% probability of detecting a 20% difference between groups (p ≤ 0.05).At their designated endpoint, the animals were euthanised and their cochleae collected for molecular and immunohistochemical analysis of cochlear inflammation. Of the two cochleae harvested from each noise-exposed mouse, one was analysed by quantitative real-time RT-PCR to determine noise-induced changes in the gene expression levels of TNF-α, IL-6, IL-1β, CCL2, and ICAM-1. Relative quantification of gene expression was determined using the comparative ΔΔCT method. The contralateral cochlea was used to determine the distribution and protein expression levels of ICAM-1 and PECAM-1 using semi-quantitative immunohistochemistry (immunoperoxidase staining). Quantitative data obtained from the study was statistically analysed using a one-way ANOVA followed by Tukey’s post-hoc multiple comparisons test (a p value of less than 0.05 was considered statistically significant).

Cochlear Inflammatory Response Associated with Chronic Noise Exposure

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Research on noise-induced hearing loss over the years has primarily been focused on the pathophysiological and functional outcomes of acute exposures to intense noise. The aim of this study was to examine the possible presence of cochlear inflammation following repeated exposure to moderate noise over an extended period of time (chronic noise exposures). C57BL/6 mice were exposed to 90 dB SPL octave-band noise (8-16 kHz) for 2 h per day for either 7 (1 week), 14 (2 weeks), 21 (3 weeks) or 28 (4 weeks) continuous days. Each experimental noise group were exposed to noise at approximately the same time each day throughout their designated noise exposure period. Animals were euthanised 24 h following the completion of the final noise exposure and their cochleae extracted. As with the previous study, quantitative real-time RT-PCR analysis was carried out to assess changes in the transcriptional expression of inflammatory mediators, and immunoperoxidase staining was performed to characterise the expression and distribution of the cell adhesion molecules ICAM-1 and PECAM-1.

Role of Adenosine A2A Receptor Signalling in Noise-Induced Cochlear Inflammation

Adenosine has potent anti-inflammatory effects in a wide range of tissues, mediated primarily via the adenosine A2A receptor. To determine its role in noise-induced cochlear inflammation, a series of interrelated studies based on adenosine A2A receptor signalling were conducted as described below.

Adenosine A2A Receptor Expression in the Noise-Exposed Cochlea

There is evidence that various forms of stress can influence the expression of adenosine receptors. In this study, the tissue distribution of the adenosine A2A receptor in the mouse cochlea and the effect of excessive noise on the expression of this receptor was investigated. Mice were exposed to acute traumatic noise (100 dB SPL, 8-16 kHz for 24 h) and their cochleae collected at 6 h, 1 day, 3 days or 7 days  after the cessation of noise exposure (n = 4 per time point). Immunofluorescence was carried out using a specific adenosine A2A receptor antibody to localise the cochlear expression and distribution of the receptor. Protein expression levels were compared qualitatively with the normal non-noise exposed cochlea.Because they are known to be abundantly expressed by a variety of immune cells, adenosine A2A receptors were also used as a marker for inflammatory cells in the noise-exposed cochlea. The total number of adenosine A2A receptor-positive infiltrating cells was counted in all cochlear turns (apical, middle and basal) of every second mid-modiolar cross-section from each noise-exposed cochlea. Labelled cells were counted under the fluorescent microscope (Nikon Eclipse 80i Microscope, Nikon Instruments Inc., Melville, NY, U.S.A.) at 40x magnification. Cochlear sections were focused throughout their entire thickness (30 µm) to ensure all infiltrating cells throughout the depth of the sections were identified. To characterise the time course of cellular infiltration, the average number of cells/section was determined in the non-exposed control cochlea and at the four time points following noise exposure.

Noise-Induced Cochlear Inflammatory Response in Adenosine A2A Receptor Knockout Mice

For the next study, the cochlear inflammatory response in noise-exposed homozygous recessive adenosine A2A receptor knockout (A2ARKO-/-) C57BL/6 mice was assessed to gain further insight into the role of adenosine A2A receptor signalling in noise-induced cochlear inflammation. A2ARKO-/- mice were exposed to acute traumatic noise (100 dB SPL, 8-16 kHz for 24 h) and euthanised at either 6 (n = 5) or 24 h (n = 5) after the cessation of the noise exposure. These time points were selected based on the results obtained from study one, in which 6 h was the initial peak time in the gene expression level of TNF-α, ICAM-1, CCL2 and IL-1β, and 24 h was the time of maximum ICAM-1 immunoexpression and cellular infiltration. The extent of cochlear inflammation was assessed by determining the protein expression level of ICAM-1 using immunoperoxidase staining and the gene expression levels of TNF-α, CCL2, ICAM-1 and IL-1β using quantitative real-time RT-PCR. It is hypothesised that a stronger cochlear inflammatory response occurs in these noise-exposed A2ARKO-/- animals. Also included in the study was a control group of A2ARKO-/- mice exposed to ambient sound levels (n = 5). The results obtained with the knockout mice were compared with those obtained from wild-type non-exposed and noise-exposed mice . The experimental groups included in this study are shown in below.

Anti-Inflammatory Effect of Regadenoson, a Selective Adenosine A2A Receptor Agonist, in the Noise-Exposed Cochlea

For this final study, the therapeutic potential of adenosine signalling in attenuating inflammatory processes in the noise-exposed cochlea was explored. This intervention was based on the selective activation of adenosine A2A receptors in the cochlea using the selective adenosine A2A receptor agonist regadenoson. Regadenoson, a FDA approved drug, is clinically used as a coronary vasodilator (pharmacological stress agent) for radionuclide myocardial perfusion imaging in patients who are unable to undergo adequate exercise stress.

1. LITERATURE REVIEW
1.1. Introduction
1.2. Cochlear Anatomy and Physiology
1.3. Inflammation
1.4. Cochlear Inflammation
1.5. Noise-Induced Hearing Loss
1.6. Noise-Induced Cochlear Inflammation
1.7. Adenosine Signalling
2. AIMS & HYPOTHESES
2.1. Aims
2.2. Hypotheses
3. METHODS
3.1 Cochlear Inflammatory Response Associated with Acute Noise Exposure
3.2 Cochlear Inflammatory Response Associated with Chronic Noise Exposure
3.3 Role of Adenosine A2A Receptor Signalling in Noise-Induced Cochlear Inflammation
3.4. Animals
3.5. Noise Exposures
3.6. Auditory Brainstem Responses (ABRs)
3.7. Cochlear Tissue Preparation
3.8. Immunohistochemistry
3.9. Quantitative Real-Time RT-PCR
3.10. Drug (Regadenoson) Preparation and Administration
3.11. Statistical Analysis
4. RESULTS
4.1. General Observations
4.2. Cochlear Inflammatory Response Associated with Acute Noise Exposure
4.3. Cochlear Inflammatory Response Associated with Chronic Noise Exposure
4.4. Role of Adenosine A2A Receptor Signalling in Noise-Induced Cochlear Inflammation
5. DISCUSSION
5.1. Introduction
5.2. Summary of Findings
5.3. Gene Expression Levels of Inflammatory Mediators in the Noise-Exposed Cochlea
5.4. Immunoexpression of Cell Adhesion Molecules in the Noise-Exposed Cochlea
5.5. Adenosine A2A Receptor Expression in the Noise-Exposed Cochlea
5.6. Adenosine A2A Receptor-Positive Infiltrating Cells in the Noise-Exposed Cochlea
5.7. Role of Noise-Induced Cochlear Inflammation
5.8. Cochlear Inflammatory Response Associated with Chronic Noise Exposure
5.9. Anti-Inflammatory Effect of Regadenoson, a Selective Adenosine A2A Receptor Agonist, in the Noise-Exposed Mouse Cochlea
5.10. Potential Therapeutic Interventions for Noise-Induced Cochlear Inflammation

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CHARACTERISATION OF NOISE-INDUCED COCHLEAR INFLAMMATION

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