Blast-Induced Tinnitus: A Combined Behavioral, Memri, And Electrophysiology Study

ABSTRACT BLAST-INDUCED TINNITUS: A COMBINED BEHAVIORAL, MEMRE, AND ELECTROPHYSIOLOGY STUDY by JESSICA OUYANG May 2014 Advisor: Drs. Steve Cala & Jinsheng Zhang Major: Physiology Degree: Doctor of Philosophy Tinnitus and hearing loss are the frequent auditory-related co-morbidities of blast trauma. The etiology of blast-induced tinnitus is also muddled by brain mechanisms associated with emotional and cognitive problems such as anxiety, memory loss, and depression. We set out to develop a realistic and ecologically valid model to address changes of cognitive status and psychological state that are associated with blast- induced tinnitus. In this study, 19 adult rats were randomly divided into the sham group (n=6) and the blast group (n=13). Blast exposure (14 psi) was conducted via a shock wave tube to expose the left ears of the rats in the blast group, and a sham exposure was conducted to the rats in the sham group. Blast-induced tinnitus was evaluated with gap detection and pre-pulse inhibition (PPI) acoustic startle reflex paradigms; the changes of thresholds of the hearing was evaluated with auditory brainstem response (ABRs), the change in the level of anxiety was evaluated with elevated plus maze; and the change in the status of memory was evaluated with one-day Morris water maze. To investigate blast-induced neuronal changes in the limbic structures, we utilized MEMRI technique. Obtained with MRIcro, MR intensity signal-to-noise ratios (SNRs) of 83 selected limbic structures were measured to represent the level of synaptic activity. Of the 13 rats that were exposed to blast shock wave, 8 rats developed chronic tinnitus on post-exposure day 35 (PED35) and 5 rats did not. Our results showed that compared to rats in the sham group (n=6), (1) rats in the blast group with or without tinnitus demonstrated higher level of anxiety (p\u3c0.05); (2) rats in the blast group that exhibited behavioral evidences of tinnitus (n=8) demonstrated neuronal hyperactivity in bilateral amygdaloidal complex, specifically bilateral basolateral groups and the left cortical-like group of the amygdala (p\u3c0.05); and (3) rats in the blast group demonstrated neuronal hyperactivity in bilateral nucleus accumbens core (p\u3c0.05). In conclusion, the elevated level of synaptic activity in the bilateral amygdala and nucleus accumbens core indicates central plasticity associated with blast-induced tinnitus

Neuroinflammation mediates noise-induced synaptic imbalance and tinnitus in rodent models

Hearing loss is a major risk factor for tinnitus, hyperacusis, and central auditory processing disorder. Although recent studies indicate that hearing loss causes neuroinflammation in the auditory pathway, the mechanisms underlying hearing loss-related pathologies are still poorly understood. We examined neuroinflammation in the auditory cortex following noise-induced hearing loss (NIHL) and its role in tinnitus in rodent models. Our results indicate that NIHL is associated with elevated expression of proinflammatory cytokines and microglial activation-two defining features of neuroinflammatory responses-in the primary auditory cortex (AI). Genetic knockout of tumor necrosis factor alpha (TNF-alpha) or pharmacologically blocking TNF-alpha expression prevented neuroinflammation and ameliorated the behavioral phenotype associated with tinnitus in mice with NIHL. Conversely, infusion of TNF-alpha into AI resulted in behavioral signs of tinnitus in both wild-type and TNF-alpha knockout mice with normal hearing. Pharmacological depletion of microglia also prevented tinnitus in mice with NIHL. At the synaptic level, the frequency of miniature excitatory synaptic currents (mEPSCs) increased and that of miniature inhibitory synaptic currents (mIPSCs) decreased in AI pyramidal neurons in animals with NIHL. This excitatory-to-inhibitory synaptic imbalance was completely prevented by pharmacological blockade of TNF-alpha expression. These results implicate neuroinflammation as a therapeutic target for treating tinnitus and other hearing loss-related disorders.National Institute of Health [DC009259, DC014335]; Department of Defense [W81XWH-15-1-0028, W81XWH-15-1-0356, W81XWH-15-1-0357]; Food and Health Bureau of Hong Kong Special Administrative Region Government [04150076]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Investigating the Role of Cortical Inhibition in Tinnitus

Subjective tinnitus is characterized as the perception of a phantom sound with no external acoustic source, and is often described as a “ringing in the ears” sensation. While evidence supports a central origin for tinnitus, the underlying neural mechanisms for this condition remain elusive. The studies presented in this thesis offer significant contributions to understanding the neural basis of tinnitus by (1) validating a behavioural paradigm that can successfully screen rats for transient noise-induced tinnitus without any indications of false-positives, and (2) demonstrating that a local loss of inhibition is sufficient to induce gain enhancement in the primary auditory cortex, as well as tinnitus-positive behaviour - evidence that supports the central gain model, one of the leading hypotheses of tinnitus generation. Overall, these findings help provide more effective strategies to directly investigate putative mechanisms of tinnitus, and furthermore expand our current understanding of this distressing condition

Terapias auditivas para acúfenos (tinnitus)

Un acúfeno (tinnitus) es la percepción de un sonido en ausencia de estimulación acústica externa, es decir, la experiencia consciente de un sonido que se origina en la propia cabeza del paciente. En colaboración con el departamento de acústica (CAEND) del Consejo Superior de Investigaciones Científicas (CSIC), se pretende revertir (de forma paliativa) las molestias, con ayuda de terapias sonoras que estimulan el sistema auditivo. Primero, se analizan los tratamientos existentes que se utilizan para atender a los pacientes diagnosticados. Por último, se diseñan dos aplicaciones informáticas referentes a las terapias: Auditory Discrimination Training (ADT) y Enriched Acoustic Environment (EAE). Abstract Tinnitus is the perception of sound in the absence of external acoustic stimulation, in addition, the conscious experience a sound originating from the patient’s own head. In collaboration with the department of acoustic (CAEND) of the Consejo Superior de Investigaciones Científicas (CSIC), is to reverse (for palliation) discomfort, using sound therapies that stimulate the auditory system. First, we analyze the existing treatments are used to treat patients diagnosed. Finally, two applications are designed regarding therapies: Auditory Discrimination Training (ADT) and Enriched Acoustic Environment (EAE)

MULTIMODAL ASSESSMENT OF CETACEAN CENTRAL NERVOUS AUDITORY PATHWAYS WITH EMPHASIS ON FORENSIC DIAGNOSTICS OF ACOUSTIC TRAUMA

Cetaceans encompass some of the world’s most enigmatic species, with one of their greatest adaptations to the marine environment being the ability to “see” by hearing. Their anatomy and behavior are fine-tuned to emit and respond to underwater sounds, which is why anthropogenic noise pollution is likely to affect them negatively. There are many effects of noise on living organisms, and while knowledge on their entire palette and interplay remain incomplete, evidence for insults ranging from acoustic trauma over behavioral changes, to masking and stress, is accumulating. Humans are subject to peak interest in terms of medical research on noise-induced hearing loss. As major health concerns can be expected across species, addressing this problem in free-ranging cetacean populations will lead to a more sustainable management of marine ecosystems, more effective and balanced policies, and successes in conservation. While progress has been made in behavioral monitoring, electrophysiological hearing assessments and post-mortem examination of the inner ear of cetaceans, but very little is known about the neurochemical baseline and neuropathology of their central auditory pathways. In the present work, we reviewed the known effects of sound on cetaceans in both wild and managed settings and explored the value of animal models of neurodegenerative disease. We began by evaluating a row of antibodies associated with neurodegeneration in a more readily available species, the dog, where acute neurological insult could be derived from clinical history. We then set out to systematically validate a key panel of protein biomarkers for the assessment of similar neurodegenerative processes of the cetacean central nervous system. For this, we developed protocols to adequately sample cetacean auditory nuclei, optimized the immunohistochemical workflow, and used Western blot and alignment of protein sequences between the antigen targeted by our antibodies and the dolphin proteome. A Histoscore was used to semi-quantitively categorize immunoreactivity patterns and dolphins by age and presence of pathology. First results indicated significant differences both between sick and healthy, and young and old animals. We then expanded our list of validated antibodies for use in the bottlenose dolphin and the techniques used to assess them in a multimodal, quantitative way. 7T-MRI and stereology were implemented to assess the neuronal, axonal, glial and fiber tract counts in the inferior colliculus and ventral cochlear nucleus of a healthy bottlenose dolphin, which created a baseline understanding of protein expression in these structures, and the influence of tissue processing. This will make a valuable comparison for when positive controls of acoustic trauma would become available. Furthermore, we explored the connectome and neuronal morphology of auditory nuclei and experimented with probe designs and machine learning algorithms to quantify structures of interest. Comparisons with pathological human brains revealed similarities in the configuration of extracellular matrix components to those of a healthy dolphin, in line with existing knowledge on the tolerance to hypoxia in these diving animals. This could have interesting implications in future investigation of the evolutionary development of marine mammal brains, as well as help diversify out-of-the-box approaches to researching human neurodegenerative disease, as is being done with hibernating species. The data and methodologies described herein contribute to the knowledge on neurochemical signature of the cetacean central nervous system. They are intended to facilitate understanding of auditory and non-auditory pathology and build an evidence-based backbone to future policies regarding noise and other form of anthropogenic threats to the marine environment.Cetaceans encompass some of the world’s most enigmatic species, with one of their greatest adaptations to the marine environment being the ability to “see” by hearing. Their anatomy and behavior are fine-tuned to emit and respond to underwater sounds, which is why anthropogenic noise pollution is likely to affect them negatively. There are many effects of noise on living organisms, and while knowledge on their entire palette and interplay remain incomplete, evidence for insults ranging from acoustic trauma over behavioral changes, to masking and stress, is accumulating. Humans are subject to peak interest in terms of medical research on noise-induced hearing loss. As major health concerns can be expected across species, addressing this problem in free-ranging cetacean populations will lead to a more sustainable management of marine ecosystems, more effective and balanced policies, and successes in conservation. While progress has been made in behavioral monitoring, electrophysiological hearing assessments and post-mortem examination of the inner ear of cetaceans, but very little is known about the neurochemical baseline and neuropathology of their central auditory pathways. In the present work, we reviewed the known effects of sound on cetaceans in both wild and managed settings and explored the value of animal models of neurodegenerative disease. We began by evaluating a row of antibodies associated with neurodegeneration in a more readily available species, the dog, where acute neurological insult could be derived from clinical history. We then set out to systematically validate a key panel of protein biomarkers for the assessment of similar neurodegenerative processes of the cetacean central nervous system. For this, we developed protocols to adequately sample cetacean auditory nuclei, optimized the immunohistochemical workflow, and used Western blot and alignment of protein sequences between the antigen targeted by our antibodies and the dolphin proteome. A Histoscore was used to semi-quantitively categorize immunoreactivity patterns and dolphins by age and presence of pathology. First results indicated significant differences both between sick and healthy, and young and old animals. We then expanded our list of validated antibodies for use in the bottlenose dolphin and the techniques used to assess them in a multimodal, quantitative way. 7T-MRI and stereology were implemented to assess the neuronal, axonal, glial and fiber tract counts in the inferior colliculus and ventral cochlear nucleus of a healthy bottlenose dolphin, which created a baseline understanding of protein expression in these structures, and the influence of tissue processing. This will make a valuable comparison for when positive controls of acoustic trauma would become available. Furthermore, we explored the connectome and neuronal morphology of auditory nuclei and experimented with probe designs and machine learning algorithms to quantify structures of interest. Comparisons with pathological human brains revealed similarities in the configuration of extracellular matrix components to those of a healthy dolphin, in line with existing knowledge on the tolerance to hypoxia in these diving animals. This could have interesting implications in future investigation of the evolutionary development of marine mammal brains, as well as help diversify out-of-the-box approaches to researching human neurodegenerative disease, as is being done with hibernating species. The data and methodologies described herein contribute to the knowledge on neurochemical signature of the cetacean central nervous system. They are intended to facilitate understanding of auditory and non-auditory pathology and build an evidence-based backbone to future policies regarding noise and other form of anthropogenic threats to the marine environment

Tinnitus and Decreased Subcortical and Cortical Inhibition

Background: The perception of tinnitus may be triggered by a reduction in inhibitory function in the central auditory nervous system. Evidence, primarily from invasive studies of animal models of tinnitus, indicates that these changes occur at both the subcortical and cortical level. Auditory evoked potential (AEP) indices of subcortical inhibition [auditory brainstem response (ABR) 〖 V/I 〗_ amp ratio ] and cortical inhibition [cortical auditory evoked potential (CAEP) sensory gating ratios] may provide an objective index of whether reduced subcortical and/or cortical inhibition is associated with tinnitus perception in humans. The aims of this study were to assess whether ABR and/or CAEP indices of subcortical and cortical inhibition distinguish between a group with constant tinnitus and matched non-tinnitus controls, and whether tinnitus presence and/or other factors [age, noise exposure history, hearing loss, speech perception in noise (SPIN)] predicted ABR and/or CAEP outcomes related to inhibition. Methods: Individuals with tinnitus and control counterparts matched for sex, age, and hearing thresholds completed the study (n = 18 per group). ABRs were recorded with a tiptrode in response to high intensity click ABRs to determine the 〖 V/I 〗_ amp ratio . CAEPs were recorded in response to two successive high intensity 10 ms clicks. A ratio of the amplitude or area of the first (conditioning CAEP) and second (test CAEP) click response was determined ( test CAEP / conditioning CAEP ) as the primary measure of sensory gating. The latency ratio was also determined as a secondary outcome which may relate to sensory gating. For both the ABR 〖 V/I 〗_ amp ratio and CAEP sensory gating ratios, a larger value indicated reduced inhibition. Ratios were compared between the two groups using independent t-tests. The relative predictive value (proportional reduction in error, PRE) of tinnitus, age, noise exposure history, hearing loss, and SPIN on ABR and CAEP outcome variables related to inhibition was analyzed using regression. Results: Individuals with tinnitus, relative to controls, exhibited similar ABR 〖 V/I 〗_ amp ratio , and significantly larger sensory gating 〖 P1 〗_ lat ratio . None of the variables assessed significantly predicted the ABR 〖 V/I 〗_ amp ratio . Tinnitus significantly predicted 〖 P1-N1 〗_ amp ratio , but not when taking into account age, noise exposure history, hearing loss, and SPIN. The 〖 P1 〗_ lat ratio was significantly predicted by both tinnitus and age, however, best predicted by age. Conclusions: Tinnitus-related reduced inhibition was not evident at the subcortical level based on the ABR 〖 V/I 〗_ amp ratio . At the cortical level, the predictive influence of tinnitus on the 〖 P1-N1 〗_ amp ratio supports the association between reduced sensory gating with tinnitus presence in humans. The significantly larger 〖 P1 〗_ lat ratio in the tinnitus group may also support reduced sensory gating and/or a change in the recovery time, or refractoriness, of auditory evoked responses in individuals with tinnitus. The strong predictive influence of age on the 〖 P1 〗_ lat ratio indicates that increasing age reduced sensory gating above and beyond the effects of tinnitus. Potential limitations to the current study, including the non-normally distributed participant characteristics and AEP methodologies, as well as considerations for future research aiming to improve the reliability and validity of tinnitus AEP assessments are discussed

Environmental and genetic risk factors for tinnitus

Tinnitus is a phantom auditory sensation, most often referred to as “ringing in the ears” with detrimental effect on quality of life. Between 4% and 37% of the global population has experienced tinnitus at some point in their life. For every 1 out of 10 individuals experiencing tinnitus, it becomes a severely impactful condition, affecting concentration, sleep, mood, and general quality of life. Despite its high prevalence and severe socio-economic burden, there is no successful treatment. The work presented in this thesis uses multiple scientific approaches to better understand the etiology of tinnitus, with the emphasis on the genetic landscape in order to gain insight into its molecular origins. First, we identify important gaps in knowledge on environmental risk factors associated with tinnitus. Second, we show using genetic epidemiology methods that severe tinnitus runs in families, which changes the current narrative that tinnitus would be generated purely due to environmental factors. Third, as tinnitus is commonly linked to hearing loss, we used a genome-wide biostatistical approach to reveal the genetic architecture of hearing loss, that will be further essential in distinguishing the two conditions. Fourth, we investigated the whole genome in relation to tinnitus to map correlated genomic regions and consequently, specific genes associated with tinnitus. Finally, we used a high-throughput sequencing of protein coding regions of the genome to identify disease-causing mutations impacting severe tinnitus. The work presented in this thesis provides insights from multiple aspects into the origins of tinnitus and will serve as a backbone to understanding the pathophysiology of the disorder

Investigations of Noise-Related Tinnitus

Tinnitus is a frequent consequence of noise trauma. Usually, however, the main focus regarding the consequences of noise trauma is placed on hearing loss, instead of tinnitus. The objectives of the present study were to assess various aspects of noise-related tinnitus in Finland, such as to determine the main causes of conscript acute acoustic traumas (AAT) in the military, assess tinnitus prevalence after noise trauma, characterize long-term AAT-related tinnitus prevalence and characteristics, assess occupational tinnitus, and evaluate the efficacy of hearing protection regulations in preventing hearing loss and tinnitus. The study comprised several independent noise-exposed groups: conscripts performing their military duty, former conscripts who suffered an AAT over a decade earlier, bomb explosion victims, and retired army personnel. Tinnitus questionnaires were used to assess tinnitus prevalence and characteristics. For occupational tinnitus, occupational noise-induced hearing loss (NIHL) reports to the Finnish Institute of Occupational Health were reviewed. Tinnitus is a common result of AAT, blast exposure and long-term noise exposure. Despite hearing protection regulations, up to hundreds of AATs occur annually among conscripts in the Finnish Defence Forces (FDF). The most common cause is an accidental shot, accounting for approximately half of the cases. Conscript AATs are mainly due to accidental shots, while the ear is unprotected. Only seldom is an AAT due to negligence. The most common causative weapon of conscript AATs is the assault rifle, accounting for 81% of conscript AATs. After AAT, the majority of tinnitus cases resolve during military service and become asymptomatic. However, in one-fifth of the cases, tinnitus persists, causing problems such as sleeping and concentration difficulties in many. In Finland, occupational tinnitus often remains unreported in conjunction with NIHL reports. In a survey of occupational NIHL cases, tinnitus was mentioned in only four per cent. However, a subsequent inquiry revealed that almost 90% in fact had tinnitus, indicating that most cases remained undetected and unreported. The best way to prevent noise-related tinnitus is prevention of noise trauma. In the military, hearing protection guidelines have been revised several times over the years. These regulations have been effective in reducing hearing loss of professional soldiers. There has also been a reduction in cases with tinnitus, but the decrease was not significant. However, with improved hearing protection regulations, a significant reduction in the risk of more serious, disturbing tinnitus was observed

Repeated low-level blast induces chronic neuroinflammation and neurobehavioral changes in rat models

Blast-induced neurotrauma (bTBI) is a signature medical concern for military personnel when they are exposed to explosions in active combat zones. However, soldiers as well as law enforcement personnel are also repeatedly exposed to low-level blasts during training sessions with heavy weaponries as part of combat readiness. Service personnel who sustain brain injuries from repeated low-level blasts (rLLBs) do not display overt pathological symptoms immediately but rather develop cognitive impairments, attention deficits, anxiety, and sleep disturbances over time. An improved rat model of rLLB was developed in this thesis by applying controlled low-level blast pressures (10 psi) repeated five times to model the true mechanism of injuries sustained by service members. Neither the etiology of rLLB nor the consequences of repeated exposure to this low-level blast are well understood. Thus, this study examined rLLB-induced acute and chronic pathological and behavioral consequences in our rat model. The first aim investigated anxiety, motor, and memory impairments at acute (1-3 days) and chronic (\u3e25 days) time points following rLLB using elevated plus maze (EPM), novel object recognition (NOR), and Rotarod tasks. Rationales for choosing these behavioral tasks were based on the literature; for example, EPM has been widely used in bTBI to assess anxiety-like symptoms, and the NOR test has shown consistent memory impairments at different BOPs whereas Morris water maze (MWM) has not. Finally, compared to other motor assessments, Rotarod has sensitivity for detecting motor coordination impairments in low-level blast. Results indicated that animals exposed to rLLB significantly displayed acute and chronic anxiety-like symptoms, motor, and short-term memory impairments compared to control (unexposed) and single low-level blast rats. The second aim explored the molecular mechanisms involved in neurobehavioral changes, including superoxide-producing NADPH oxidase (NOX1), microglial activation, and reactive astrocytosis as likely contributing factors. Of the many pathological mechanisms present following brain injury, chronic neuroinflammation has been observed for up to 17 years post-TBI and has neuroinflammation been observed even months after bTBI. Chronic persistent neuroinflammation can induce neurotoxicity. Microglia are the innate immune cells of the central nervous system and have both beneficial and detrimental effects based on their activation period. Chronic microglial activation causes continued release of free radicals, cytokines (IL-1β, TNF-α), chemokines, and other signaling molecules, resulting in neurobehavioral and pathological changes. Microglial-mediated behavioral deficits can be caused by proinflammatory cytokine IL-1β. Studies have shown that excessive IL-1β levels have an influence on anxiety, motor, and short-term recognition memory impairments in clinical and preclinical research. A molecular mechanism that is involved in IL-1β release is the inflammasome complex, particularly NLRP3. NOX1-mediated oxidative stress and NLRP3 -mediated IL-1β release have not been investigated in rLLB. This aim explored the role of the NLRP3 inflammasome complex in regulating caspase-1 activation and subsequent release of the proinflammatory cytokines IL-1β in chronic neuroinflammation. Using immunofluorescence, this study examined NOX1 and NLRP3 expression, microglial activation, and astrocytosis using specific primary antibodies such as NOX1, NLRP3, Iba-1, and GFAP, respectively in the hippocampus due to its high susceptibility for blast wave compared with any other brain region. Results indicated that there was an increase NOX1 and NLRP3 protein expression, microglial activation, and reactive astrocytosis following rLLB The third aim investigated the therapeutic effects of MCC950, a specific NLRP3 inhibitor, on neurobehavioral and neuropathological abnormalities following rLLB. This study mainly examines MCC950\u27s protective role against rLLB-induced chronic microglial activation and inflammation, especially NLRP3-IL-1β-mediated neurobehavioral changes. This study performed single immunofluorescence to assess microglial activation and double immunofluorescence followed by colocalization analysis to evaluate NLRP3+ microglia in the hippocampus and perirhinal cortex following rLLB. Treatment with MCC950 prevented short-term recognition memory impairments and mitigated NLRP3 and cleaved caspase-1 expression and IL-1β release. Furthermore, inhibition of the NLRP3 inflammasome by administration of MCC950 displayed an improvement in behavioral and pathological changes caused by rLLB, validating the original hypothesis. Therefore, targeting microglial activation by inhibiting the NLRP3 inflammasome activation may have a therapeutic potential to counteract rLLB-induced chronic neurobehavioral changes