Automated High-Throughput Damage Scoring of Zebrafish Lateral Line Hair Cells After Ototoxin Exposure.

Zebrafish have emerged as a powerful biological system for drug development against hearing loss. Zebrafish hair cells, contained within neuromasts along the lateral line, can be damaged with exposure to ototoxins, and therefore, pre-exposure to potentially otoprotective compounds can be a means of identifying promising new drug candidates. Unfortunately, anatomical assays of hair cell damage are typically low-throughput and labor intensive, requiring trained experts to manually score hair cell damage in fluorescence or confocal images. To enhance throughput and consistency, our group has developed an automated damage-scoring algorithm based on machine-learning techniques that produce accurate damage scores, eliminate potential operator bias, provide more fidelity in determining damage scores that are between two levels, and deliver consistent results in a fraction of the time required for manual analysis. The system has been validated against trained experts using linear regression, hypothesis testing, and the Pearson's correlation coefficient. Furthermore, performance has been quantified by measuring mean absolute error for each image and the time taken to automatically compute damage scores. Coupling automated analysis of zebrafish hair cell damage to behavioral assays for ototoxicity produces a novel drug discovery platform for rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

A Fully Automated High-Throughput Zebrafish Behavioral Ototoxicity Assay.

Zebrafish animal models lend themselves to behavioral assays that can facilitate rapid screening of ototoxic, otoprotective, and otoregenerative drugs. Structurally similar to human inner ear hair cells, the mechanosensory hair cells on their lateral line allow the zebrafish to sense water flow and orient head-to-current in a behavior called rheotaxis. This rheotaxis behavior deteriorates in a dose-dependent manner with increased exposure to the ototoxin cisplatin, thereby establishing itself as an excellent biomarker for anatomic damage to lateral line hair cells. Building on work by our group and others, we have built a new, fully automated high-throughput behavioral assay system that uses automated image analysis techniques to quantify rheotaxis behavior. This novel system consists of a custom-designed swimming apparatus and imaging system consisting of network-controlled Raspberry Pi microcomputers capturing infrared video. Automated analysis techniques detect individual zebrafish, compute their orientation, and quantify the rheotaxis behavior of a zebrafish test population, producing a powerful, high-throughput behavioral assay. Using our fully automated biological assay to test a standardized ototoxic dose of cisplatin against varying doses of compounds that protect or regenerate hair cells may facilitate rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

Older Individuals Meeting Medicare Cochlear Implant Candidacy Criteria in Noise but Not in Quiet: Are These Patients Improved by Surgery?

OBJECTIVE: To investigate postoperative hearing outcomes in older patients who qualified for cochlear implant (CI) by Medicare criteria using AZBio sentence tests performed in noise but not in quiet. STUDY DESIGN: Review of patient records. SETTING: University-based otology/neurotology practice. PATIENTS: The senior author performed 136 CI between January 2013 and September 2015. Starting in 2013, CI candidacy evaluation included AZBio sentence tests performed in quiet and noise. For the current study, older patients with preoperative AZBio scores greater than 40% in quiet but less than 40% in noise (+10 or +5 dB signal to noise ratio [SNR]) and follow up >/=6 months were included. INTERVENTION(S): Cochlear implantation in one ear. MAIN OUTCOME MEASURE(S): Pre- versus postoperative AZBio sentence test scores. RESULTS: Fifteen patients with an average age of 73 years (range, 59-91) met inclusion criteria. Preoperative AZBio scores for the implanted ear averaged 47% points in quiet and 9% points in noise (+10 or +5 dB SNR). Preoperative bilateral AZBio scores averaged 70% points in quiet and 24% points in noise (+10 or +5 dB SNR). Postoperative AZBio scores for the implanted ear improved an average of 71% points in quiet and 51% points in noise. Postoperative bilateral hearing improved 23% points in quiet and 27% points in noise. CONCLUSION: All patients undergoing CI candidacy testing should be tested in both quiet and noise conditions. For those who qualify only in noise, our results demonstrate that cochlear implantation typically improves hearing both in quiet and noise.

Evaluating the control: minipump implantation and breathing behavior in the neonatal rat.

We evaluated genioglossus (GG) gross motoneuron morphology, electromyographic (EMG) activities, and respiratory patterning in rat pups allowed to develop without interference (unexposed) and pups born to dams subjected to osmotic minipump implantation in utero (saline-exposed). In experiment 1, 48 Sprague-Dawley rat pups (Charles-River Laboratories), ages postnatal day 7 (P7) through postnatal day 10 (P10), were drawn from two experimental groups, saline-exposed (n = 24) and unexposed (n = 24), and studied on P7, P8, P9, or P10. Pups in both groups were sedated (Inactin hydrate, 70 mg/kg), and fine-wire electrodes were inserted into the GG muscle of the tongue and intercostal muscles to record EMG activities during breathing in air and at three levels of normoxic hypercapnia [inspired CO2 fraction (FiCO2 ): 0.03, 0.06, and 0.09]. Using this approach, we assessed breathing frequency, heart rate, apnea type, respiratory event types, and respiratory stability. In experiment 2, 16 rat pups were drawn from the same experimental groups, saline-exposed (n = 9) and unexposed (n = 7), and used in motoneuron-labeling studies. In these pups a retrograde dye was injected into the GG muscle, and the brain stems were subsequently harvested and sliced. Labeled GG motoneurons were identified with microscopy, impaled, and filled with Lucifer yellow. Double-labeled motoneurons were reconstructed, and the number of primary projections and soma volumes were calculated. Whereas pups in each group exhibited the same number (P = 0.226) and duration (P = 0.093) of respiratory event types and comparable motoneuron morphologies, pups in the implant group exhibited more central apneas and respiratory instability relative to pups allowed to develop without interference.

The Case for Using Evidence-Based Guidelines in Setting Hospital and Public Health Policy.

OBJECTIVE: Hospital systems and regulating agencies enforce strict guidelines barring personal items from entering the operating room (OR) - touting surgical site infections (SSIs) and patient safety as the rationale. We sought to determine whether or not evidence supporting this recommendation exists by reviewing available literature. BACKGROUND DATA: Rules and guidelines that are not evidence based may lead to increased hospital expenses and limitations on healthcare provider autonomy. METHODS: PubMed, Embase, Scopus, Cochrane Library, Web of Science, and CINAHL were searched in order to find articles that correlated personal items in the OR to documented SSIs. Articles that satisfied the following criteria were included: (1) studies looking at personal items in the OR, such as handbags, purses, badges, pagers, backpacks, jewelry phones, and eyeglasses, but not just OR equipment; and (2) the primary outcome measure was infection at the surgical site. RESULTS: Seventeen articles met inclusion criteria and were evaluated. Of the 17, the majority did not determine if personal items increased risk for SSIs. Only one article examined the correlation between a personal item near the operative site and SSI, concluding that wedding rings worn in the OR had no impact on SSIs. Most studies examined colonization rates on personal items as potential infection risk; however, no personal items were causally linked to SSI in any of these studies. CONCLUSION: There is no objective evidence to suggest that personal items in the OR increase risk for SSIs.

Zebrafish swimming behavior as a biomarker for ototoxicity-induced hair cell damage: a high-throughput drug development platform targeting hearing loss.

Hearing loss is one of the most common human sensory disabilities, adversely affecting communication, socialization, mood, physical functioning, and quality of life. In addition to age and noise-induced damage, ototoxicity is a common cause of sensorineural hearing loss with chemotherapeutic agents, for example, cisplatin, being a major contributor. Zebrafish (Danio rerio) are an excellent model to study hearing loss as they have neurosensory hair cells on their body surface that are structurally similar to those within the human inner ear. Anatomic assays of toxin-mediated hair cell damage in zebrafish have been established; however, using fish swimming behavior--rheotaxis--as a biomarker for this anatomic damage was only recently described. We hypothesized that, in parallel, multilane measurements of rheotaxis could be used to create a high-throughput platform for drug development assessing both ototoxic and potentially otoprotective compounds in real time. Such a device was created, and results demonstrated a clear dose response between cisplatin exposure, progressive hair cell damage, and reduced rheotaxis in zebrafish. Furthermore, pre-exposure to the otoprotective medication dexamethasone, before cisplatin exposure, partially rescued rheotaxis swimming behavior and hair cell integrity. These results provide the first evidence that rescued swimming behavior can serve as a biomarker for rescued hair cell function. Developing a drug against hearing loss represents an unmet clinical need with global implications. Because hearing loss from diverse etiologies may result from common end-effects at the hair cell level, lessons learned from the present study may be broadly used.

Developmental nicotine exposure adversely effects respiratory patterning in the barbiturate anesthetized neonatal rat.

Neonates at risk for sudden infant death syndrome (SIDS) are hospitalized for cardiorespiratory monitoring however, monitoring is costly and generates large quantities of averaged data that serve as poor predictors of infant risk. In this study we used a traditional autocorrelation function (ACF) testing its suitability as a tool to detect subtle alterations in respiratory patterning in vivo. We applied the ACF to chest wall motion tracings obtained from rat pups in the period corresponding to the mid-to-end of the third trimester of human pregnancy. Pups were drawn from two groups: nicotine-exposed and saline-exposed at each age (i.e., P7, P8, P9, and P10). Respiratory-related motions of the chest wall were recorded in room air and in response to an arousal stimulus (FIO2 14%). The autocorrelation function was used to determine measures of breathing rate and respiratory patterning. Unlike alternative tools such as Poincare plots that depict an averaged difference in a measure breath to breath, the ACF when applied to a digitized chest wall trace yields an instantaneous sample of data points that can be used to compare (data) points at the same time in the next breath or in any subsequent number of breaths. The moment-to-moment evaluation of chest wall motion detected subtle differences in respiratory pattern in rat pups exposed to nicotine in utero and aged matched saline-exposed peers. The ACF can be applied online as well as to existing data sets and requires comparatively short sampling windows (∼2 min). As shown here, the ACF could be used to identify factors that precipitate or minimize instability and thus, offers a quantitative measure of risk in vulnerable populations.

Neural drive to respiratory muscles in the spontaneously breathing rat pup.

The neonatal rodent serves as useful and appropriate model within which to study respiratory system development. Despite an extensive literature that documents respiratory control in vitro, in vivo studies have relied upon whole body plethysmography to determine measures of respiratory frequency and tidal volume. However, plethysmography restricts access to the animal and thus, respiratory muscle electromyographic (EMG) activities have not been recorded in these studies previously. Electromyography yields accurate information about neural respiratory center output to the musculature and therefore, about the control of breathing in the intact animal. In this case, we documented neural drive to respiratory pump and upper airway muscles, electrocardiogram (ECG) and chest wall motions in rat pups up to 10 days of age noting sighs, spontaneous central apneas and hypopneas in room air and with successive increments in fractional inspired CO2 (FICO2). Our findings underscore the advantages of EMG recordings for purposes of determining the magnitude and distribution of neural drive to respiratory muscles and for characterizing the full range of breathing behaviors exhibited by rats in the early postnatal period.