Using an appetitive operant conditioning paradigm to screen rats for tinnitus induced by intense sound exposure: Experimental considerations and interpretation.

In an effort to help elucidate the neural mechanisms underlying tinnitus in humans, researchers have often relied on animal models; a preclinical approach which ultimately required that behavioral paradigms be designed to reliably screen animals for tinnitus. Previously, we developed a two-alternative forced-choice (2AFC) paradigm for rats that allowed for the simultaneous recording of neural activity at the very moments when they were reporting the presence/absence of tinnitus. Because we first validated our paradigm in rats experiencing transient tinnitus following a high-dose of sodium salicylate, the present study now sought to evaluate its utility to screen for tinnitus caused by intense sound exposure; a common tinnitus-inducer in humans. More specifically, through a series of experimental protocols, we aimed to (1) conduct sham experiments to ensure that the paradigm was able to correctly classify control rats as not having tinnitus, (2) confirm the time course over which the behavioral testing could reliably be performed post-exposure to assess chronic tinnitus, and (3) determine if the paradigm was sensitive to the variable outcomes often observed after intense sound exposure (e.g., hearing loss with our without tinnitus). Ultimately, in accordance with our predictions, the 2AFC paradigm was indeed resistant to false-positive screening of rats for intense sound-induced tinnitus, and it was able to reveal variable tinnitus and hearing loss profiles in individual rats following intense sound exposure. Taken together, the present study documents the utility of our appetitive operant conditioning paradigm to assess acute and chronic sound-induced tinnitus in rats. Finally, based on our findings, we discuss important experimental considerations that will help ensure that our paradigm is able to provide a suitable platform for future investigations into the neural basis of tinnitus.

Noise exposure in early adulthood causes age-dependent and brain region-specific impairments in cognitive function.

Hearing loss is a chronic health condition that affects millions of people worldwide. In addition to age-related hearing impairment, excessive noise exposure is a leading cause of hearing loss. Beyond the devastating effects of hearing impairment itself, epidemiological studies have identified hearing loss as a major risk factor for age-related cognitive decline, including dementia. At present, we currently lack a full understanding of the brain regions and underlying molecular changes that are responsible for mediating the link between hearing loss and cognitive impairment across aging. In the present study, we exposed 6-month-old rats to an occupational-like noise (100 dB SPL, 4 h/day × 30 days) or sham exposure and investigated both hippocampal-dependent (i.e., spatial learning and memory, assessed using the Morris water maze) and striatal-dependent (i.e., visuomotor associative learning, assessed using an operant-conditioning task) cognitive function across aging at 7, 10, and 13 months of age. We also investigated brain region-specific changes in microglial expression following noise/sham exposure in order to assess the potential contribution of this cell type to noise-induced cognitive impairments. Consistent with human studies, the occupational-like noise exposure resulted in high-frequency hearing loss, evidenced by a significant increase in hearing thresholds at 20 kHz. Ultimately, our results suggest that not all higher-level cognitive tasks or their associated brain regions appear to be equally susceptible to noise-induced deficits during aging, as the occupational-like noise exposure caused an age-dependent deficit in spatial but not visuomotor associative learning, as well as altered microglial expression in the hippocampus but not the striatum. Interestingly, we found no significant relationships between spatial learning ability and the level of hearing loss or altered microglial density in the hippocampus following noise exposure, suggesting that other changes in the brain likely contribute to hippocampal-dependent cognitive dysfunction following noise exposure. Lastly, we found that a subset of younger animals also showed noise-induced deficits in spatial learning; findings which suggest that noise exposure may represent an increased risk for cognitive impairment in vulnerable subjects. Overall, our findings highlight that even a mild occupational-like noise exposure earlier in adulthood can have long lasting implications for cognitive function later in life.

Brain Targeting and Toxicological Assessment of the Extracellular Vesicle-Packaged Antioxidant Catalase-SKL Following Intranasal Administration in Mice.

The antioxidant enzyme catalase represents an important therapeutic target due to its role in mitigating cellular reactive oxygen species that contribute to the pathogenesis of many disease states. Catalase-SKL (CAT-SKL), a genetically engineered, peroxisome-targeted, catalase derivative, was developed in order to increase the therapeutic potential of the enzyme, and has previously been shown to be effective in combating oxidative stress in a variety of in vitro and in vivo models, thereby mitigating cellular degeneration and death. In the present study we addressed important considerations for the development of an extracellular vesicle-packaged version of CAT-SKL (evCAT-SKL) as a therapeutic for neurodegenerative diseases by investigating its delivery potential to the brain when administered intranasally, and safety by assessing off-target toxicity in a mouse model. Mice received weekly intranasal administrations of evCAT-SKL or empty extracellular vesicles for 4 weeks. Fluorescent labeling for CAT-SKL was observed throughout all sections of the brain in evCAT-SKL-treated mice, but not in empty extracellular vesicle-treated mice. Furthermore, we found no evidence of gross or histological abnormalities following evCAT-SKL or empty extracellular vesicle treatment in a full-body toxicological analysis. Combined, the successful brain targeting and the lack of off-target toxicity demonstrates that intranasal delivery of extracellular vesicle-packaged CAT-SKL holds promise as a therapeutic for addressing neurological disorders.

Uncovering the contribution of enhanced central gain and altered cortical oscillations to tinnitus generation.

Various theories and their associated mechanisms have been proposed as the neural basis of phantom sound perception (tinnitus), including central gain enhancement and altered cortical oscillations. However, it remains unknown whether these cortical changes directly cause tinnitus, or simply coexist with the phantom percept. Using chronically-implanted electrodes and drug delivery cannulae in rats, we examined whether enhanced central gain and cortical oscillations are consistent across different tinnitus induction methods (noise exposure; salicylate), and if directly-inducing enhanced central gain or altered cortical oscillations via pharmacologic manipulation of inhibition along the auditory pathway would cause behavioral evidence of tinnitus. We show that, while there appeared to be no clear link between tinnitus and the presence of enhanced sound-evoked cortical activity or altered spontaneous cortical oscillations, pharmacologic impairment of GABAergic neurotransmission in the auditory cortex was sufficient to cause tinnitus; collective findings which further advance our understanding of the neural basis of tinnitus.

Differential Plasticity in Auditory and Prefrontal Cortices, and Cognitive-Behavioral Deficits Following Noise-Induced Hearing Loss.

Excessive exposure to loud noise causes hearing loss and neural plasticity throughout the auditory pathway. Recent studies have identified that non-auditory regions, such as the hippocampus, are also susceptible to noise exposure; however, the electrophysiological and behavioral consequences of noise-induced hearing loss on the prefrontal cortex (PFC) are unclear. Using chronically-implanted electrodes in awake rats, we investigated neural plasticity in the auditory and prefrontal cortices in the days following noise exposure via metrics associated with spontaneous neural oscillations and the 40-Hz auditory steady-state response (ASSR). Noise exposure did not alter the profile of spontaneous oscillations in either of the cortices, yet it caused a differential plasticity in the sound-evoked activity, which was characterized by enhanced event-related potentials (ERPs) in the auditory cortex (i.e., central gain), and decreased inter-trial coherence (ITC) of the 40-Hz ASSR within the PFC. Moreover, phase synchrony between auditory and prefrontal cortices was decreased post-exposure, suggesting a reduction in functional connectivity. Cognitive-behavioral testing using the Morris water maze and a series of lever-pressing tasks revealed that noise exposure impaired spatial learning and reference memory, as well as stimulus-response habit learning, whereas cognitive flexibility tasks requiring set-shifting and reversal learning appeared unaffected. Collectively, our findings identify the complex and region-specific cortical plasticity associated with noise-induced hearing loss, and highlight the varying degrees of susceptibility of non-auditory, cognitive tasks of learning, memory and executive function to noise exposure.

Noise-induced hearing loss alters hippocampal glucocorticoid receptor expression in rats.

Although the effects of intense noise exposure on the peripheral and central auditory pathway have been well characterized, its effects on non-classical auditory structures in the brain, such as the hippocampus, are less well understood. Previously, we demonstrated that noise-induced hearing loss causes a significant long-term reduction in hippocampal neurogenesis and cell proliferation. Given the known suppressive effects of stress hormones on neurogenesis, the goal of the present study was to determine if activation of the stress response is an underlying mechanism for the long-term reduction in hippocampal neurogenesis observed following noise trauma. To accomplish this, we monitored basal and reactive blood plasma levels of the stress hormone corticosterone in rats for ten weeks following acoustic trauma, and quantified changes in hippocampal glucocorticoid and mineralocorticoid receptors. Our results indicate that long-term auditory deprivation does not cause a persistent increase in basal or reactive stress hormone levels in the weeks following noise exposure. Instead, we observed a greater decline in reactive corticosterone release in noise-exposed rats between the first and tenth week of sampling compared to control rats. We also observed a significant increase in hippocampal glucocorticoid receptor expression which may cause greater hippocampal sensitivity to circulating glucocorticoid levels and result in glucocorticoid-induced suppression of neurogenesis, as well as increased feedback inhibition on the HPA axis. No change in mineralocorticoid receptor expression was observed between control and noise exposed rats. These results highlight the adverse effect of intense noise exposure and auditory deprivation on the hippocampus.

Low-cost blast wave generator for studies of hearing loss and brain injury: blast wave effects in closed spaces.

BACKGROUND: Military personnel and civilians living in areas of armed conflict have increased risk of exposure to blast overpressures that can cause significant hearing loss and/or brain injury. The equipment used to simulate comparable blast overpressures in animal models within laboratory settings is typically very large and prohibitively expensive. NEW METHOD: To overcome the fiscal and space limitations introduced by previously reported blast wave generators, we developed a compact, low-cost blast wave generator to investigate the effects of blast exposures on the auditory system and brain. RESULTS: The blast wave generator was constructed largely from off the shelf components, and reliably produced blasts with peak sound pressures of up to 198dB SPL (159.3kPa) that were qualitatively similar to those produced from muzzle blasts or explosions. Exposure of adult rats to 3 blasts of 188dB peak SPL (50.4kPa) resulted in significant loss of cochlear hair cells, reduced outer hair cell function and a decrease in neurogenesis in the hippocampus. COMPARISON TO EXISTING METHODS: Existing blast wave generators are typically large, expensive, and are not commercially available. The blast wave generator reported here provides a low-cost method of generating blast waves in a typical laboratory setting. CONCLUSIONS: This compact blast wave generator provides scientists with a low cost device for investigating the biological mechanisms involved in blast wave injury to the rodent cochlea and brain that may model many of the damaging effects sustained by military personnel and civilians exposed to intense blasts.

Behavioral models of tinnitus and hyperacusis in animals.

The phantom perception of tinnitus and reduced sound-level tolerance associated with hyperacusis have a high comorbidity and can be debilitating conditions for which there are no widely accepted treatments. One factor limiting the development of treatments for tinnitus and hyperacusis is the lack of reliable animal behavioral models of these disorders. Therefore, the purpose of this review is to highlight the current animal models of tinnitus and hyperacusis, and to detail the advantages and disadvantages of each paradigm. To date, this is the first review to include models of both tinnitus and hyperacusis.

A novel behavioral assay for the assessment of acute tinnitus in rats optimized for simultaneous recording of oscillatory neural activity.

BACKGROUND: Human magneto/electrophysiology studies suggest that the phantom sound of tinnitus arises from spontaneous oscillatory neural activity in auditory cortex; however, in animal models, behavioral techniques suitable for testing this hypothesis in combination with electrophysiology recordings have yet to be evaluated. While electrophysiological studies of tinnitus have been reported in passive, awake animals, these studies fail to control for attentional mechanisms likely to play a role in the perception of tinnitus. NEW METHOD: A novel appetitive operant conditioning, two-alternative identification task was developed for detecting acute tinnitus in rats. The procedure optimizes conditions for simultaneously recording oscillatory neural activity while controlling for the attentional state of the animal. RESULTS: Tinnitus was detected in six of seven rats following systemic injection with sodium salicylate (200mg/kg IP), a known inducer of tinnitus. Analysis of ongoing local field potentials recorded from chronically implanted electrodes in auditory cortex of a rat reporting tinnitus revealed changes in the spectrum of ongoing neural activity. Comparison with existing method(s): Existing tinnitus-detection methods were not explicitly designed for the simultaneous recording of neural activity. The behavioral method reported here is the first to provide the conditions necessary for obtaining these recordings in chronically implanted rats. CONCLUSIONS: The behavioral assay presented here will facilitate research into the neural mechanisms of tinnitus by allowing researchers to compare the electrophysiological data in animals with confirmed tinnitus.

The gap-startle paradigm for tinnitus screening in animal models: limitations and optimization.

In 2006, Turner and colleagues (Behav. Neurosci., 120:188-195) introduced the gap-startle paradigm as a high-throughput method for tinnitus screening in rats. Under this paradigm, gap detection ability was assessed by determining the level of inhibition of the acoustic startle reflex produced by a short silent gap inserted in an otherwise continuous background sound prior to a loud startling stimulus. Animals with tinnitus were expected to show impaired gap detection ability (i.e., lack of inhibition of the acoustic startle reflex) if the background sound containing the gap was qualitatively similar to the tinnitus pitch. Thus, for the gap-startle paradigm to be a valid tool to screen for tinnitus, a robust startle response from which to inhibit must be present. Because recent studies have demonstrated that the acoustic startle reflex could be dramatically reduced following noise exposure, we endeavored to 1) modify the gap-startle paradigm to be more resilient in the presence of hearing loss, and 2) evaluate whether a reduction in startle reactivity could confound the interpretation of gap prepulse inhibition and lead to errors in screening for tinnitus. In the first experiment, the traditional broadband noise (BBN) startle stimulus was replaced by a bandpass noise in which the sound energy was concentrated in the lower frequencies (5-10 kHz) in order to maintain audibility of the startle stimulus after unilateral high-frequency noise exposure (16 kHz). However, rats still showed a 57% reduction in startle amplitude to the bandpass noise post-noise exposure. A follow-up experiment on a separate group of rats with transiently-induced conductive hearing loss revealed that startle reactivity was better preserved when the BBN startle stimulus was replaced by a rapid airpuff to the back of the rat's neck. Furthermore, it was found that transient unilateral conductive hearing loss, which was not likely to induce tinnitus, caused an impairment in gap prepulse inhibition as assessed with the traditional BBN gap-startle paradigm, resulting in a false-positive screening for tinnitus. Thus, the present study identifies significant caveats of the traditional gap-startle paradigm, and describes experimental parameters using an airpuff startle stimulus which may help to limit the negative consequences of reduced startle reactivity following noise exposure, thereby allowing researchers to better screen for tinnitus in animals with hearing loss.

Lithium chloride regulates the proliferation of stem-like cells in retinoblastoma cell lines: a potential role for the canonical Wnt signaling pathway.

PURPOSE: Cancer stem cells are found in many tumor types and are believed to lead to regrowth of tumor mass due to their chemoresistance and self-renewal capacity. We previously demonstrated small subpopulations of cells in retinoblastoma tissue and cell lines that display cancer stem cell-like activities, including expression of stem cell markers, Hoechst dye exclusion, slow cycling, and self-renewal ability. Identifying factors regulating stem cell proliferation will be important for selectively targeting stem cells and controlling tumor growth. Wingless and Int1 (Wnt) signaling is an essential cellular communication pathway that regulates proliferation and differentiation of non-neoplastic stem/progenitor cells in the retina and other tissues, but its role in cancer stem cells in the retinal tumor retinoblastoma is unknown. In this study, we investigated whether the Wnt pathway activator lithium chloride (LiCl) regulates proliferation of retinoblastoma cancer stem-like cells. METHODS: The number of stem-like cells in Weri and Y79 retinoblastoma cell line cultures was measured by 5-bromo-2-deoxyuridine (BrdU) pulse-chase, immunohistochemistry, and quantitative polymerase chain reaction (PCR) for stem cell marker genes. The cell lines were sorted into stem-like and non-stem-like populations by fluorescence-activated cell sorting (FACS), using an antibody against the stem cell marker ATP-binding cassette, subfamily G, member 2 (ABCG2). Activated Wnt signaling was measured in the sorted cells by western blotting and immunolocalization of the central mediator beta-catenin. RESULTS: LiCl increased the number of stem-like cells, measured by BrdU retention and elevated expression of the stem cell marker genes Nanog, octamer transcription factor 3 and 4 (Oct3/4), Musashi 1 (Msi1), and ABCG2. Sorted ABCG2-positive stem-like cells had higher levels of beta-catenin than ABCG2-negative non-stem cells, suggesting elevated canonical Wnt signaling. Furthermore, stem cell marker gene expression increased after small interfering RNA (siRNA) knock-down of the Wnt inhibitor secreted frizzled-related protein 2 (SFRP2). CONCLUSIONS: These results indicate that the cancer stem-like cell population in retinoblastoma is regulated by canonical Wnt/beta-catenin signaling, which identifies the Wnt pathway as a potential mechanism for the control of stem cell renewal and tumor formation in retinoblastoma tumors in vivo.

Immunoreactivity of ICAM-1 in human tumors, metastases and normal tissues.

Intercellular adhesion molecule-1 (ICAM-1) is implicated to play a role in cancer metastasis, and may serve as a diagnostic tool for tumor prognosis and progression as well as a target for therapeutic intervention. The aim of this study was to carry out a comprehensive survey of ICAM-1 immunoreactivity in normal, malignant and metastatic tissues. We assessed immunoreactivity of ICAM-1 in a total of 300 tissue cores from multiple tissue arrays of normal, malignant, and metastatic tissues by immunohistochemistry. We scored tissue samples for ICAM-1 immunoreactivity on a 0-3 scale, assessed the number of samples exhibiting infiltrating immune cells, and documented ICAM-1 immunoreactivity in some specific cell types. ICAM-1 expression in normal tissues was highest in spleen and absent in the cerebrum, peripheral nerves, pancreas, ovary, breast, uterus, cervix, prostate, lung, larynx, bone marrow, striated muscle, heart, mesothelium, esophagus, small intestine, colon and liver. In primary malignancies, lymphoid tissues received the highest average ICAM-1 score while connective tissue/skin had the lowest average ICAM-1 score. Of the metastatic tissues, those originating from the urinary tract had the highest average ICAM-1 score while those originating from glandular tissues had the lowest average ICAM-1 score. Metastases localized in lymphoid tissues had a higher average ICAM-1 score than those localized in non-lymphoid tissues. Since ICAM-1 is associated with a variety of cancer types and appears to play a role in cancer metastasis, our findings should serve as a helpful resource for investigations of ICAM-1 as a biomarker, as well as a target for therapeutic interventions.