Faculty Bibliography

Balance and movement are impaired in many neurological disorders. Recent advances in behavioral monitoring provide unprecedented access to posture and locomotor kinematics but without the throughput and scalability necessary to screen candidate genes/potential therapeutics. Here, we present a scalable apparatus to measure posture and locomotion (SAMPL). SAMPL includes extensible hardware and open-source software with real-time processing and can acquire data from D. melanogaster, C. elegans, and D. rerio as they move vertically. Using SAMPL, we define how zebrafish balance as they navigate vertically and discover small but systematic variations among kinematic parameters between genetic backgrounds. We demonstrate SAMPL's ability to resolve differences in posture and navigation as a function of effect size and data gathered, providing key data for screens. SAMPL is therefore both a tool to model balance and locomotor disorders and an exemplar of how to scale apparatus to support screens.

Chronic pain involves sensitization of nociceptors and synaptic transmission of painful signals in nociceptive circuits in the dorsal horn of the spinal cord. We investigated the contribution of clathrin-dependent endocytosis to sensitization of nociceptors by G protein-coupled receptors (GPCRs) and to synaptic transmission in spinal nociceptive circuits. We determined whether therapeutic targeting of endocytosis could ameliorate pain. mRNA encoding dynamin (Dnm) 1-3 and adaptor-associated protein kinase 1 (AAK1), which mediate clathrin-dependent endocytosis, were localized to primary sensory neurons of dorsal root ganglia of mouse and human and to spinal neurons in the dorsal horn of the mouse spinal cord by RNAScope®. When injected intrathecally to mice, Dnm and AAK1 siRNA or shRNA knocked-down Dnm and AAK1 mRNA in dorsal root ganglia neurons, reversed mechanical and thermal allodynia and hyperalgesia, and normalized non-evoked behavior in preclinical models of inflammatory and neuropathic pain. Intrathecally administered inhibiters of clathrin, Dnm and AAK1 also reversed allodynia and hyperalgesia. Disruption of clathrin, Dnm and AAK1 did not affect normal motor functions of behaviors. Patch clamp recordings of dorsal horn neurons revealed that Dnm1 and AAK1 disruption inhibited synaptic transmission between primary sensory neurons and neurons in lamina I/II of the spinal cord dorsal horn by suppressing release of synaptic vesicles from presynaptic primary afferent neurons. Patch clamp recordings from dorsal root ganglion nociceptors indicated that Dnm siRNA prevented sustained GPCR-mediated sensitization of nociceptors. By disrupting synaptic transmission in the spinal cord and blunting sensitization of nociceptors, endocytosis inhibitors offer a therapeutic approach for pain treatment.

Systemic symptoms resulting from exposure to members of the Lepidoptera order, such as moths, butterflies, and caterpillars, are known as lepidopterism. Most cases of lepidopterism are mild and result from dermal exposure to urticating hairs; ingestion is less common and generally more medically significant because the hairs may get embedded in the patient's mouth, hypopharynx, or esophagus, leading to dysphagia, drooling, edema, and possible airway obstruction. In previous cases of symptomatic caterpillar ingestion reported in the literature, extensive efforts, including direct laryngoscopy, esophagoscopy, and bronchoscopy, were undertaken to remove these hairs. We review the case of a 19-mo-old previously healthy male infant who presented to the emergency department with vomiting and inconsolability after ingesting half of a woolly bear caterpillar (Pyrrharctia isabella). His initial examination was notable for embedded hairs in his lips, oral mucosa, and right tonsillar pillar. The patient underwent a bedside flexible laryngoscopy, which revealed a single hair embedded in the epiglottis, without significant edema. He was stable from a respiratory standpoint and, thus, was admitted for observation and IV dexamethasone without any attempt to remove the hairs. He was discharged in good condition after 48 h; at a follow-up visit 1 wk later, no remaining hairs were visible. This case demonstrates that lepidopterism secondary to caterpillar ingestion is amenable to conservative management and does not require routine removal of urticating hairs in patients who do not show signs of airway distress.

Vestibulospinal neurons integrate sensed imbalance to regulate postural reflexes. As an evolutionarily conserved neural population, understanding their synaptic and circuit-level properties can offer insight into vertebrate antigravity reflexes. Motivated by recent work, we set out to verify and extend the characterization of vestibulospinal neurons in the larval zebrafish. Using current-clamp recordings together with stimulation, we observed that larval zebrafish vestibulospinal neurons are silent at rest, yet capable of sustained spiking following depolarization. Neurons responded systematically to a vestibular stimulus (translation in the dark); responses were abolished after chronic or acute loss of the utricular otolith. Voltage-clamp recordings at rest revealed strong excitatory inputs with a characteristic multimodal distribution of amplitudes, as well as strong inhibitory inputs. Excitatory inputs within a particular mode (amplitude range) routinely violated refractory period criteria and exhibited complex sensory tuning, suggesting a nonunitary origin. Next, using a unilateral loss-of-function approach, we characterized the source of vestibular inputs to vestibulospinal neurons from each ear. We observed systematic loss of high-amplitude excitatory inputs after utricular lesions ipsilateral, but not contralateral, to the recorded vestibulospinal neuron. In contrast, while some neurons had decreased inhibitory inputs after either ipsilateral or contralateral lesions, there were no systematic changes across the population of recorded neurons. We conclude that imbalance sensed by the utricular otolith shapes the responses of larval zebrafish vestibulospinal neurons through both excitatory and inhibitory inputs. Our findings expand our understanding of how a vertebrate model, the larval zebrafish, might use vestibulospinal input to stabilize posture. More broadly, when compared with recordings in other vertebrates, our data speak to conserved origins of vestibulospinal synaptic input.

BACKGROUND/OBJECTIVES/OBJECTIVE:Base of tongue (BOT) dysfunction is common following oropharyngeal concurrent chemoradiation therapy (CCRT). We present a clinically relevant animal model quantifying the effects of CCRT on tongue strength and elasticity over time. METHODS:Fifty-three male and 53 female Sprague-Dawley rats were randomized to control or experimental groups. Experimental animals received cisplatin, 5-fluorouracil, and 5 fractions of 7 Gy directed to the BOT. Controls received no intervention. At 2 weeks, 5 months, or 10 months after CCRT, animals underwent non-survival surgery to measure twitch and tetanic tongue strength, which were analyzed using multivariate linear mixed effects models. Tongue displacement, a surrogate for tongue elasticity, was also determined via stress-strain testing and analyzed via a multivariate linear mixed effects model. RESULTS:Reporting the combined results of both sexes, the estimated experimental group mean peak twitch forces became more divergent over time compared to controls, being 8.3% lower than controls at 2 weeks post-CCRT, 15.7% lower at 5 months, and 31.6% lower at 10 months. Estimated experimental group mean peak tetanic forces followed a similar course and were 2.9% lower than controls at 2 weeks post CCRT, 20.7% lower at 5 months, and 27.0% lower at 10 months. Stress-strain testing did not find CCRT to have a significant effect on tongue displacement across experimental timepoints. CONCLUSIONS:This study demonstrates an increasing difference in tongue strength over time between controls and animals exposed to CCRT. Tongue elasticity was not significantly affected by CCRT, suggesting that changes in strength may not be caused by fibrosis. LEVEL OF EVIDENCE/METHODS:NA Laryngoscope, 2022.

OBJECTIVE:To compare treatment outcomes for T4b head and neck adenoid cystic carcinoma (ACC). STUDY DESIGN/METHODS:Historical cohort study. SETTING/METHODS:National Cancer Database (NCDB). METHODS:Identified all T4b ACC of head and neck origin diagnosed 2004 to 2019 in the NCDB. Demographics, clinical characteristics, treatment details, and survival were analyzed. Treatment outcomes were analyzed using univariable and multivariable Cox regression. RESULTS:We identified 606 cases of T4b ACC. Less than half (284, 47.0%) underwent curative-intent treatment. Among these, most were treated with primary surgery: surgery + radiotherapy (RT) (122, 43.0%) or surgery + chemoradiotherapy (CRT) (42, 14.8%). The positive margin rate was 78.7%, and 90-day postoperative mortality was zero. Nonsurgical patients were treated with definitive RT (60, 21.1%) or definitive CRT (60, 21.1%). The median follow-up was 51.5 months. Overall survival was 77.8% at 3 years. Three-year survival was higher for patients treated with surgery compared to those treated nonsurgically (84% vs 70%; p = .005). Surgical treatment remained associated with higher survival on multivariable analysis (hazard ratio [HR]: 0.47, p = .005). This effect was most pronounced for oral cavity tumors (HR: 0.17, p = .01). Among matched cohorts of surgically treated patients, there was no difference in 3-year survival between clinical T4a and T4b tumors (83.3% vs 83.0%, p = .99). CONCLUSION/CONCLUSIONS:Long-term survival for T4b ACC of the head and neck could be expected. Primary surgical treatments can be performed safely and are associated with longer survival. A carefully selected subset of patients with very advanced ACC might benefit from the consideration of surgical treatments.

The hypothesis that sustained G protein-coupled receptor (GPCR) signaling from endosomes mediates pain is based on studies with endocytosis inhibitors and lipid-conjugated or nanoparticle-encapsulated antagonists targeted to endosomes. GPCR antagonists that reverse sustained endosomal signaling and nociception are needed. However, the criteria for rational design of such compounds are ill-defined. Moreover, the role of natural GPCR variants, which exhibit aberrant signaling and endosomal trafficking, in maintaining pain is unknown. Herein, substance P (SP) was found to evoke clathrin-mediated assembly of endosomal signaling complexes comprising neurokinin 1 receptor (NK₁R), Gα_q/i, and βarrestin-2. Whereas the FDA-approved NK₁R antagonist aprepitant induced a transient disruption of endosomal signals, analogs of netupitant designed to penetrate membranes and persist in acidic endosomes through altered lipophilicity and pKa caused sustained inhibition of endosomal signals. When injected intrathecally to target spinal NK₁R+ve neurons in knockin mice expressing human NK₁R, aprepitant transiently inhibited nociceptive responses to intraplantar injection of capsaicin. Conversely, netupitant analogs had more potent, efficacious, and sustained antinociceptive effects. Mice expressing C-terminally truncated human NK₁R, corresponding to a natural variant with aberrant signaling and trafficking, displayed attenuated SP-evoked excitation of spinal neurons and blunted nociceptive responses to SP. Thus, sustained antagonism of the NK₁R in endosomes correlates with long-lasting antinociception, and domains within the C-terminus of the NK₁R are necessary for the full pronociceptive actions of SP. The results support the hypothesis that endosomal signaling of GPCRs mediates nociception and provides insight into strategies for antagonizing GPCRs in intracellular locations for the treatment of diverse diseases.

OBJECTIVE:Glucocorticoids (GCs) modulate multiple cellular activities including inflammatory and fibrotic responses. Outcomes of GC treatment for laryngeal disease vary, affording opportunity to optimize treatment. In the current study, three clinically employed GCs were evaluated to identify optimal in vitro concentrations at which GCs mediate favorable anti-inflammatory and fibrotic effects in multiple cell types. We hypothesize a therapeutic window will emerge as a foundation for optimized therapeutic strategies for patients with laryngeal disease. STUDY DESIGN/METHODS:In vitro. METHODS:to alter inflammatory and fibrotic gene expression was calculated. RESULTS:to downregulate other genes. CONCLUSION/CONCLUSIONS:Lower concentrations of GCs repressed inflammatory gene expression and only moderately induced genes involved in fibrosis. These data warrant consideration as a foundation for optimized clinical care paradigms to reduce inflammation and mitigate fibrosis. LEVEL OF EVIDENCE/METHODS:NA Laryngoscope, 133:1169-1175, 2023.