Subacute acrolein exposure to rat larynx in vivo.

OBJECTIVES/HYPOTHESIS: Inhaled pollutants can contact vocal fold tissue and induce detrimental voice changes. Acrolein is a pollutant in cigarette smoke and can also be inhaled during the combustion of fossil fuels, animal fats, and plastics in the environment. However, the vocal fold pathological changes induced by acrolein and the underlying inflammatory pathways are not well understood. These biologic data are needed to understand why voice problems may result from pollutant exposure. STUDY DESIGN: In vivo prospective design with experimental and control groups. METHODS: Sprague-Dawley male rats (N = 36) were exposed to acrolein (3 ppm) or filtered air (control) through a whole-body exposure system for 5 hours/day, for 5 days/week, over 4 weeks. Histopathological changes, presence of edema, expression of proinflammatory cytokines and markers, and the phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were investigated. RESULTS: Histological evaluation and quantification demonstrated that subacute acrolein exposure induced significant vocal fold edema. Acrolein exposure also induced epithelial sloughing and cell death. Quantitative polymerase chain reaction showed a significant upregulation of genes encoding interferon regulatory factor and chitinase-3-like protein 3. Western blot revealed a 76.8% increase in phosphorylation of NF-κB P65 after subacute acrolein exposure. CONCLUSIONS: These findings suggest that 4-week exposures to 3 ppm acrolein induce vocal fold inflammation manifested as edema, related to the activation of NF-κB signaling. The edema may underlie the voice changes reported in speakers exposed to pollutants. LEVEL OF EVIDENCE: NA Laryngoscope, 129:E313-E317, 2019.

Cigarette Smoke Exposure to Pig Larynx in an Inhalation Chamber.

OBJECTIVES: This study investigated the effects of cigarette smoke exposure on the pig larynx using an inhalation chamber. Specifically, we compared the effects of cigarette smoke exposure from either 3 cigarettes per day (3cd) or 15 cigarettes per day (15cd) for 20 days. STUDY DESIGN: In vivo prospective design. METHODS: Female pigs were exposed via an inhalation chamber to cigarette smoke (3R4F research cigarettes) from 3cd (n = 6) or 15cd (n = 6) for 20 days. Outcomes included histopathology of vocal fold and airway tissues; gene expression of interleukins, TNF-α, and VEGF; protein levels of TNF-α and IL-6; and number of coughs recorded in the chamber. RESULTS: Pigs exposed to cigarette smoke from 15cd exhibited mild vocal fold edema as compared to the 3cd group on histopathological evaluation. There was also minimal inflammation of nasal and tracheal tissue characterized by presence of more granulocytes in the 15cd group compared to the 3cd group. Cough frequency was significantly greater for the 15cd group compared to the 3cd group. CONCLUSIONS: A custom-designed large animal inhalation chamber successfully challenged pigs repeatedly, to varying levels of cigarette smoke. Future studies will combine such low levels of smoke exposure with other common challenges such as acid reflux to understand the multifactorial causation of laryngeal pathologies.

Listeria Adhesion Protein Induces Intestinal Epithelial Barrier Dysfunction for Bacterial Translocation.

Intestinal epithelial cells are the first line of defense against enteric pathogens, yet bacterial pathogens, such as Listeria monocytogenes, can breach this barrier. We show that Listeria adhesion protein (LAP) induces intestinal epithelial barrier dysfunction to promote bacterial translocation. These disruptions are attributed to the production of pro-inflammatory cytokines TNF-α and IL-6, which is observed in mice challenged with WT and isogenic strains lacking the surface invasion protein Internalin A (ΔinlA), but not a lap- mutant. Additionally, upon engagement of its surface receptor Hsp60, LAP activates canonical NF-κB signaling, facilitating myosin light-chain kinase (MLCK)-mediated opening of the epithelial barrier via cellular redistribution of the epithelial junctional proteins claudin-1, occludin, and E-cadherin. Pharmacological inhibition of MLCK or NF-κB in cells or genetic ablation of MLCK in mice prevents mislocalization of junctional proteins and L. monocytogenes translocation. Thus, L. monocytogenes uses LAP to exploit epithelial defenses and cross the intestinal epithelial barrier.

Cholesterol Esterification Inhibition Suppresses Prostate Cancer Metastasis by Impairing the Wnt/ß-catenin Pathway.

Dysregulation of cholesterol is a common characteristic of human cancers including prostate cancer. This study observed an aberrant accumulation of cholesteryl ester in metastatic lesions using Raman spectroscopic analysis of lipid droplets in human prostate cancer patient tissues. Inhibition of cholesterol esterification in prostate cancer cells significantly suppresses the development and growth of metastatic cancer lesions in both orthotopic and intracardiac injection mouse models. Gene expression profiling reveals that cholesteryl ester depletion suppresses the metastatic potential through upregulation of multiple regulators that negatively impact metastasis. In addition, Wnt/ß-catenin, a vital pathway for metastasis, is downregulated upon cholesteryl ester depletion. Mechanistically, inhibition of cholesterol esterification significantly blocks secretion of Wnt3a through reduction of monounsaturated fatty acid levels, which limits Wnt3a acylation. These results collectively validate cholesterol esterification as a novel metabolic target for treating metastatic prostate cancer. Mol Cancer Res; 16(6); 974-85. ©2018 AACR.

Early pathological characterization of murine dissecting abdominal aortic aneurysms.

We report here on the early pathology of a well-established murine model of dissecting abdominal aortic aneurysms (AAAs). Continuous infusion of angiotensin II (AngII) into apolipoprotein E-deficient mice induces the formation of aortic dissection and expansion at some point after implantation of miniosmotic pumps containing AngII. While this model has been studied extensively at a chronic stage, we investigated the early pathology of dissecting AAA formation at multiple scales. Using high-frequency ultrasound, we screened 12-week-old male mice daily for initial formation of these aneurysmal lesions between days 3 and 10 post-implantation. We euthanized animals on the day of diagnosis of a dissecting AAA or at day 10 if no aneurysmal lesion developed. Aortic expansion and reduced vessel wall strain occurred in animals regardless of whether a dissecting AAA developed by day 10. The aortas of mice that did not develop dissecting AAAs showed intermediate changes in morphology and biomechanical properties. RNA sequencing and gene expression analysis revealed multiple proinflammatory and matrix remodeling genes to be upregulated in the suprarenal aorta of AngII-infused mice as compared to saline-infused controls. Histology and immunohistochemistry confirmed that extracellular matrix remodeling and inflammatory cell infiltration, notably neutrophils and macrophages, occurred in AngII-infused mice with and without dissecting AAAs but not saline-infused controls. Understanding early disease processes is a critical step forward in translating experimental results in cardiovascular disease research. This work advances our understanding of this well-established murine model with applications for improving early diagnosis and therapy of acute aortic syndrome in humans.

Proton density-weighted laryngeal magnetic resonance imaging in systemically dehydrated rats.

OBJECTIVES/HYPOTHESIS: Dehydrated vocal folds are inefficient sound generators. Although systemic dehydration of the body is believed to induce vocal fold dehydration, this causative relationship has not been demonstrated in vivo. Here we investigate the feasibility of using in vivo proton density (PD)-weighted magnetic resonance imaging (MRI) to demonstrate hydration changes in vocal fold tissue following systemic dehydration in rats. STUDY DESIGN: Animal study. METHODS: Sprague-Dawley rats (n = 10) were imaged at baseline and following a 10% reduction in body weight secondary to withholding water. In vivo, high-field (7 T), PD-weighted MRI was used to successfully resolve vocal fold and salivary gland tissue structures. RESULTS: Normalized signal intensities within the vocal fold decreased postdehydration by an average of 11.38% ± 3.95% (mean ± standard error of the mean [SEM], P = .0098) as compared to predehydration levels. The salivary glands experienced a similar decrease in normalized signal intensity by an average of 10.74% ± 4.14% (mean ± SEM, P = .0195) following dehydration. The correlation coefficient (percent change from dehydration) between vocal folds and salivary glands was 0.7145 (P = .0202). CONCLUSIONS: Ten percent systemic dehydration induced vocal fold dehydration as assessed by PD-weighted MRI. Changes in the hydration state of vocal fold tissue were highly correlated with that of the salivary glands in dehydrated rats in vivo. These preliminary findings demonstrate the feasibility of using PD-weighted MRI to quantify hydration states of the vocal folds and lay the foundation for further studies that explore more routine and realistic magnitudes of systemic dehydration and rehydration. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E222-E227, 2018.

Murine ultrasound-guided transabdominal para-aortic injections of self-assembling type I collagen oligomers.

Abdominal aortic aneurysms (AAAs) represent a potentially life-threatening condition that predominantly affects the infrarenal aorta. Several preclinical murine models that mimic the human condition have been developed and are now widely used to investigate AAA pathogenesis. Cell- or pharmaceutical-based therapeutics designed to prevent AAA expansion are currently being evaluated with these animal models, but more minimally invasive strategies for delivery could improve their clinical translation. The purpose of this study was to investigate the use of self-assembling type I collagen oligomers as an injectable therapeutic delivery vehicle in mice. Here we show the success and reliability of a para-aortic, ultrasound-guided technique for injecting quickly-polymerizing collagen oligomer solutions into mice to form a collagen-fibril matrix at body temperature. A commonly used infrarenal mouse AAA model was used to determine the target location of these collagen injections. Ultrasound-guided, closed-abdominal injections supported consistent delivery of collagen to the area surrounding the infrarenal abdominal aorta halfway between the right renal artery and aortic trifurcation into the iliac and tail arteries. This minimally invasive approach yielded outcomes similar to open-abdominal injections into the same region. Histological analysis on tissue removed on day 14 post-operatively showed minimal in vivo degradation of the self-assembled fibrillar collagen and the majority of implants experienced minimal inflammation and cell invasion, further confirming this material's potential as a method for delivering therapeutics. Finally, we showed that the typical length and position of this infrarenal AAA model was statistically similar to the length and targeted location of the injected collagen, increasing its feasibility as a localized therapeutic delivery vehicle. Future preclinical and clinical studies are needed to determine if specific therapeutics incorporated into the self-assembling type I collagen matrix described here can be delivered near the aorta and locally limit AAA expansion.