Evidence of Key Tinnitus-Related Brain Regions Documented by a Unique Combination of Manganese-Enhanced MRI and Acoustic Startle Reflex Testing

Animal models continue to improve our understanding of tinnitus pathogenesis and aid in development of new treatments. However, there are no diagnostic biomarkers for tinnitus-related pathophysiology for use in awake, freely moving animals. To address this disparity, two complementary methods were combined to examine reliable tinnitus models (rats repeatedly administered salicylate or exposed to a single noise event): inhibition of acoustic startle and manganese-enhanced MRI. Salicylate-induced tinnitus resulted in wide spread supernormal manganese uptake compared to noise-induced tinnitus. Neither model demonstrated significant differences in the auditory cortex. Only in the dorsal cortex of the inferior colliculus (DCIC) did both models exhibit supernormal uptake. Therefore, abnormal membrane depolarization in the DCIC appears to be important in tinnitus-mediated activity. Our results provide the foundation for future studies correlating the severity and longevity of tinnitus with hearing loss and neuronal activity in specific brain regions and tools for evaluating treatment efficacy across paradigms

Successful treatment of Enterocytozoon bieneusi gastrointestinal infection with nitazoxanide in a immunocompetent patient

Microsporidia are organisms that are known to cause opportunistic infections in immunocompromised individuals. The gastrointestinal tract is the most common affected organ. Enterocytozoon bieneusi is the most common species that infect humans. There are no known established guidelines for the treatment of this particular microsporidium. A 72-year old immunocompetent female presented to our hospital with diarrhea for four weeks. She had failed outpatient oral antimicrobial treatment for suspected traveler’s diarrhea and Clostridium difficile. Initial stool cultures were negative but given her persistent symptomatology, stool PCR was sent to rule out microsporidia and was positive for Enterocytozoon bieneusi. Patient failed treatment with albendazole. She was then subsequently treated with nitazoxanide and achieved successful infection resolution. This case demonstrates the importance of considering atypical infections in patient with persistent symptoms and suggest that nitazoxanide is effective in treating infection caused by Enterocytozoon bieneusi microsporidia. Keywords: Enterocytozoon bieneusi, Microsporidia, diarrhea, nitazoxanid

Summary graph of gap ASR comparing experimental groups (control, salicylate, noise) at three different time points (baseline, post treatment 1 and post treatment 2).

<p>For control animals the percent inhibition of startle remains constant. For the salicylate group post treatment 1 testing resulted in decreased inhibition of gASR, but by post treatment 2 testing, gASR had returned to normal levels. The 10 kHz noise group demonstrated a decreased ability to inhibit gASR at both time points following noise exposure when compared to baseline. Error bars equal SEM.</p

Summary of hearing thresholds following salicylate or noise treatment: Auditory brainstem responses.

a<p>Significance p≤0.05.</p>b<p>Hearing threshold, expressed in decibels (dB).</p>c<p>Error (±) is expressed as SEM.</p><p>100↑More than 100 dB.</p><p>Bold indicates the protected (plugged) ear (Noise I left ear protected Noise II had the right ear protected).</p

Hearing thresholds at two time points following salicylate administration: Auditory brainstem responses.

a<p>Hearing threshold, expressed in decibels (dB).</p>b<p>Error (±) is expressed as SEM.</p

Salicyate increases MEMRI measured signal intensity in the DCN but not the VCN.

<p>In the dorsal cochlear nucleus (DCN) a significant increase in signal intensity was observed (<b>A–B</b>) in the salicylate treated group (242. 37±12.08; p = 0.02) when compared to controls (210.26±6.76), but no change was observed in the VCN (<b>A, C</b>). Noise exposure does not change MEMRI measured signal intensity in the DCN (209. 90±5.22; p = 0.02) or VCN (187. 41±9.02; p = 0.02) 48 hrs post noise exposure (A–C). Signal intensity is calculated as a percentage of nearby muscle. Asterisk denotes significance, p≤0.05; error bars equal StDev, C – control, S – salicylate, N - noise exposed In MRI panels white arrows indicate VCN, green arrows indicate DCN, and blue arrows indicate ventricular space.</p

Time line showing day and approximate time of day for each procedure.

<p>Acoustic startle reflex testing (ASR) for tinnitus perception, auditory brainstem response testing (ABR) for hearing loss, tinnitus induction, manganese injection and imaging in control (<b>A</b>), salicylate (<b>B</b>) and noise (<b>C</b>) groups during a given day. 8 – first eight hrs of day, 16 – second eight hr period of day, 24 – last eight hr period of day, SA – salicylate injection, NE – noise exposure, Mn²⁺ - manganese injection, MRI – magnetic resonance imaging.</p

Effects of salicylate and noise exposure on neuronal activity in different brain regions as measured by manganese enhanced MRI.

a<p>Bold text indicates beginning of new brain region.</p>b<p>Signal intensity, expressed as the percentage of adjacent muscle; arbitrary units (a.u.).</p>c<p>Significance p≤0.05.</p>d<p>Error (±) is expressed as SEM.</p

Salicylate results in a decreased ability to blunt the acoustic startle response (ASR) under conditions of gap inhibition.

<p>Performance during ASR testing was recorded before treatment (Baseline), 1 hr following the second day of salicylate administration (Post Treatment 1) and seven hrs following the third day of salicylate administration (Post Treatment 2). Relative to a full startle response (100%), untreated rats detected the gap and prepulse and could suppress the startle reflex across all frequencies (baseline in <b>A</b>). Across all frequencies significant decreases in pASR were observed relative to baseline both one hour following salicylate administration (Post Treatment 1) and seven hours after treatment (Post Treatment 2) (<b>A</b>). When compared to baseline, p = 0.0001 for both Post Treatment 1 and 2 (compare a–b and a–c). Post Treatment 1 and 2 were also significantly different from one another (b–c; p = 0.0004). At 12 kHz the % sound off startle initially changed by 12% (p = 0.05) from Baseline to Post Treatment 1, but was not significantly different from Baseline during Post Treatment 2 testing (<b>B</b>). Differing letters denote significance across time points, p≤0.05 was significant; error bars equal SEM.</p

Effects of salicyate and noise exposure on MEMRI measured signal intensity in the inferior colliculus after three days of salicylate administration and 48 hrs post noise exposure.

<p>Signal intensity was compared across groups (A–D). In the salicylate group, manganese accumulation was supernormal in the central nucleus of the inferior colliculus (B; CNIC; 174. 20±3.60; p = 0.03), as well as the dorsal cortex (C; DCIC; 195. 21±4.96; p = 0.005) and external cortex (D; ECIC; 186. 52±2.35; p = 0.02) when compared to controls. In the noise exposed group, only the DCIC had increased signal intensity (186. 35±2.17; p = 0.03). Asterisks denote significance, p≤0.05; error bars equal StDev, C – control, S – salicylate, N - noise exposed.</p