In Vivo Visualization and Quantification of Rat Laryngeal Blood Supply After Hydration Challenge.

OBJECTIVES: Systemic dehydration decreases total body blood volume; however, hemodynamic alterations at the level of local organs, such as the larynx, remain unclear. Here we sought to quantify superior thyroid artery (STA) blood flow after dehydration and rehydration using in vivo magnetic resonance angiography (MRA) and ultrasound imaging in a rat model. METHODS: Male Sprague-Dawley rats (N = 17) were included in this prospective, repeated measures design. Rats first underwent MRA to determine baseline STA cross-sectional area, followed by high-frequency in vivo ultrasound imaging to measure STA blood velocity at baseline. Next, rats were systemically dehydrated (water withholding), followed by rehydration (water ad-lib). Ultrasound imaging was repeated immediately after dehydration and following rehydration. The STA blood velocity and STA cross-sectional area were used to compute STA blood flow. Three rats served as temporal controls for ultrasound imaging. To determine if the challenges to hydration status affected the STA cross-sectional area, four rats underwent only MRA at baseline, dehydration, and rehydration. RESULTS: Systemic dehydration resulted in 10.5% average body weight loss. Rehydration resulted in average body weight gain of 10.9%. Statistically significant reductions were observed in STA mean blood flow rate after dehydration. Rehydration reversed these changes to pre-dehydration levels. No significant differences were observed in STA cross-sectional area with dehydration or rehydration. CONCLUSION: Systemic dehydration decreased blood flow in the superior thyroid artery. Rehydration restored blood flow in the STA. Change in hydration status did not alter the STA cross-sectional area. These preliminary findings demonstrate the feasibility of using ultrasound and MRA to quantify hemodynamic changes and visualize laryngeal blood vessels. LEVEL OF EVIDENCE: NA Laryngoscope, 134:779-785, 2024.

Hydration State and Hyaluronidase Treatment Significantly Affect Porcine Vocal Fold Biomechanics.

OBJECTIVES: The understanding of vocal fold hydration state, including dehydrated, euhydrated, rehydrated tissue, and how hydration affects vocal fold biomechanical properties is still evolving. Although clinical observations support the benefits of increasing vocal fold hydration after dehydrating events, more mechanistic information on the effects of vocal fold dehydration and the beneficial effects of rehydration are needed. Alterations to hyaluronic acid (HA), an important component of the vocal fold extracellular matrix, are likely to influence the biomechanical properties of vocal folds. In this study, we investigated the influence of hydration state and HA on vocal fold tissue stiffness via biomechanical testing. STUDY DESIGN: Prospective, ex vivo study design. METHODS: Fresh porcine vocal folds (N = 18) were examined following sequential immersion in hypertonic (dehydration) and isotonic solutions (rehydration). In a separate experiment, vocal folds were incubated in hyaluronidase (Hyal) to remove HA. Control tissues were not exposed to any challenges. A custom micromechanical system with a microforce sensing probe was used to measure the force-displacement response. Optical strain was calculated, and ultrasound imaging was used to measure tissue cross-sectional area to obtain stress-strain curves. RESULTS: Significant increases (P ≤ 0.05) were found in tangent moduli between dehydrated and rehydrated vocal folds at strains of ε = 0.15. The tangent moduli of Hyal-digested tissues significantly increased at both ε = 0.15 and 0.3 (P ≤ 0.05). CONCLUSION: Vocal fold dehydration increased tissue stiffness and rehydration reduced the stiffness. Loss of HA increased vocal fold stiffness, suggesting a potential mechanical role for HA in euhydrated vocal folds.

The role of systemic dehydration in vocal fold healing: Preliminary findings.

Rationale: Systemic dehydration negatively alters the expression of vocal fold inflammatory and cell junction markers. These biological changes can have downstream effects on the healing processes of injured vocal folds. In the dermis, reduced hydration prolongs inflammation and delays healing. It is unknown whether this biological effect is observed in vocal fold tissue. Objective: To investigate the effects of systemic dehydration on vocal fold healing outcomes following acute, bilateral vocal fold injury in a rodent model. Methods: Eighteen systemic dehydrated and 18 euhydrated adult male Sprague Dawley rats experienced bilateral vocal fold injuries or no injury (N = 9/group). Vocal fold gene expression levels of inflammatory mediators and epithelial cell junction markers were measured 24 h post-injury. Results: Pro-inflammatory gene markers (IL-1ß; TNF-α) were differentially expressed in response to systemic dehydration with vocal fold injury compared to non-injury. Epithelial cell junction markers (Cadherin-3, Desmoglein-1) also exhibited divergent trends following systemic dehydration, but these data were not statistically significant. Conclusions: Systemic dehydration may affect cellular vocal fold healing processes within 24 h. These findings lay the groundwork for further investigation of how hydration status can affect vocal fold tissue recovery and influence clinical care.

Proteomic analysis reveals that aging rabbit vocal folds are more vulnerable to changes caused by systemic dehydration.

BACKGROUND: Older adults are more prone to develop systemic dehydration. Systemic dehydration has implications for vocal fold biology by affecting gene and protein expression. The objective of this study was to quantify vocal fold protein changes between two age groups and hydration status, and to investigate the interaction of age and hydration status on protein expression, which has not been investigated in the context of vocal folds before. Comparative proteomics was used to analyze the vocal fold proteome of 6.5-month-old and > 3-year-old rabbits subjected to water ad libitum or water volume restriction protocol. RESULTS: Young and older adult rabbits (n = 22) were either euhydrated (water ad libitum) or dehydrated by water volume restriction. Dehydration was confirmed by body weight loss of - 5.4% and - 4.6% in young and older groups, respectively, and a 1.7-fold increase of kidney renin gene expression in the young rabbits. LC-MS/MS identified 2286 proteins in the rabbit vocal folds of young and older adult rabbits combined. Of these, 177, 169, and 81 proteins were significantly (p ≤ 0.05) affected by age, hydration status, or the interaction of both factors, respectively. Analysis of the interaction effect revealed 32 proteins with opposite change patterns after dehydration between older and young rabbit vocal folds, while 31 proteins were differentially regulated only in the older adult rabbits and ten only in the young rabbits in response to systemic dehydration. The magnitude of changes for either up or downregulated proteins was higher in the older rabbits. These proteins are predominantly related to structural components of the extracellular matrix and muscle layer, suggesting a disturbance in the viscoelastic properties of aging vocal fold tissue, especially when subjected to systemic dehydration. CONCLUSIONS: Water restriction is a laboratory protocol to assess systemic dehydration-related changes in the vocal fold tissue that is translatable to human subjects. Our findings showed a higher number of proteins differentially regulated with a greater magnitude of change in the vocal folds of older adult rabbits in the presence of systemic dehydration compared to younger rabbits. The association of these proteins with vocal fold structure and biomechanical properties suggests that older human subjects may be more vulnerable to the effects of systemic dehydration on vocal function. The clinical implications of these protein changes warrant more investigation, but age should be taken into consideration when evaluating vocal treatment recommendations that interfere with body fluid balance.

Restricted Water Intake Adversely Affects Rat Vocal Fold Biology.

OBJECTIVES: A holistic understanding of the many ways that systemic dehydration affects vocal fold biology is still evolving. There are also myriad physiologically relevant methodologies to induce systemic dehydration. To untangle the effects of systemic dehydration on vocal fold biology, we need to utilize realistic, clinically translatable paradigms of systemic dehydration in lab animals. Restricted access to water accommodates clinical translation. We investigated whether systemic dehydration via reduced water intake would negatively affect vocal fold biology. STUDY DESIGN: Prospective, in vivo study design. METHODS: Male Sprague Dawley rats (N = 13) were provided 4 mL/100 g of water/day for 5 days, whereas male control rats (N = 8) were given ad lib access to water. Following euthanasia, tissues were processed for histological staining, gene expression, and protein assays. RESULTS: Renin gene expression level in kidneys increased significantly (P ≤ .05), validating dehydration. Dehydration induced by restricted water access downregulated the gene expression of interleukin-1α and desmoglein-1 (P ≤ .05). Hyaluronidase-2 gene expression increased after dehydration (P ≤ .05). The protein level of desmoglein-1 decreased after dehydration (P ≤ .05). Histological analyses suggested decreased hyaluronan (P ≤ .05) in the water-restricted rat vocal fold. CONCLUSION: Reduced daily water intake for just 5 days impairs vocal fold biology by disrupting inflammatory cytokine release, reducing plasma membrane integrity, and disrupting the hyaluronan network. This is the first study investigating the dehydrating effects of restricted water intake on vocal fold tissue in an in vivo model. LEVEL OF EVIDENCE: NA (prospective animal study). Laryngoscope, 131:839-845, 2021.

Genome-wide identification of lncRNAs as novel prognosis biomarkers of glioma.

BACKGROUND: Glioma is the primary cancer of the central nervous system, and defining the prognosis of glioma is of great significance in the clinical. The long noncoding RNAs (lncRNAs) emerge as important regulators of pathological processes. This study aimed to identify lncRNAs which could function as potential prognosis biomarkers of glioma. MATERIAL AND METHODS: Glioma RNA-seq data from TCGA and CGGA were analyzed to identify neoplasm grade associated lncRNAs by DEseq. 2R and weighted gene co-expression network analysis. Consensus module genes were analyzed in Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway to predict lncRNAs biological functions. Then neutrophil immune estimations were analyzed by Tumor Immune Estimation Resource. Transcrption factors of these lncRNAs were predicted by PROMO. Overall survival and receiver operating characteristic (ROC) analyses were applied to test the accuracy of predicted lncRNAs as the markers of prognosis. RESULTS: We identified four lncRNAs most correlated with both higher neoplasm grade and worse prognosis, including AC064875.2, HOTAIRM1, LINC00908, and RP11-84A19.3. Neutrophil-mediated immunity and cell adhesion junction were considered as the main biological functions of these lncRNAs. In addition, the correlation of these four lncRNAs with glioma prognosis was validated. CONCLUSION: Neutrophil immune infiltration is implicated in higher neoplasm grade and worse prognosis of glioma. AC064875.2, HOTAIRM1, LINC00908, and RP11-84A19.3 may serve as potential prognosis biomarkers of glioma.

Prevalence and Prognostic Significance of HPV in Laryngeal Squamous Cell Carcinoma in Northeast China.

BACKGROUND/AIMS: Human papillomavirus (HPV) is an etiological risk factor for a subset of head and neck squamous cell carcinomas. HPV has been proven to be a powerful prognostic biomarker for oropharyngeal cancer, but its role in the larynx has not been explored in depth. Here, we sought to evaluate the prevalence and genotype distribution of HPV in patients with laryngeal squamous cell carcinoma (LSCC) in northeast China. METHODS: HPV DNA in specimens from 211 patients diagnosed with LSCC was analyzed by the polymerase chain reaction and in situ hybridization, and p16 overexpression was evaluated by immunohistochemistry. p16 expression was scored positive if strong and diffuse nuclear and cytoplasmic staining was present in > 75% of tumor cells. RESULTS: In this study, infection with HPV and p16 expression were not absolutely consistent. Among all patients, 132 (62.6%) were positive for HPV DNA (HPV+), while 23 (10.9%) were inconsistent for HPV and p16. Multivariate analysis indicated that HPV, but not p16, is an independent prognostic factor for overall survival in LSCC. Overall survival was significantly improved in HPV+ LSCC patients compared with the HPV-negative group (hazard ratio, 0.395; 95% confidence interval, 0.185-0.843; p = 0.016). Among the 132 HPV+ patients, 28 (21.2%) were HPV-16 single infection. CONCLUSION: This study indicates that HPV DNA is a more reliable surrogate marker than p16 for the prediction of survival in patients with LSCC.

Up-regulation of Vps4A promotes neuronal apoptosis after intracerebral hemorrhage in adult rats.

Vps4, vacuolar protein sorting 4, belongs to ATPases Associated with diverse cellular Activities (AAA) protein family which is made up of Vps4A and Vps4B. Previous studies demonstrated that Vps4A plays vital roles in diverse aspects such as virus budding, the efficient transport of H-Ras to the PM (plasma membrane) and the involvement in the MVB (multivesiculate bodies) pathway. Interestingly, Vps4A is also expressed in the brain. However, the distribution and function of Vps4A in ICH diseases remain unclear. In this study, we show that Vps4A may be involved in neuronal apoptosis during pathophysiological processes of intracerebral hemorrhage (ICH). Based on the results of Western blot and immunohistochemistry, we found a remarkable up-regulation of Vps4A expression surrounding the hematoma after ICH. Double labeled immunofluorescence showed that Vps4A was co-expressed with NeuN but rarely with astrocytes and microglia. Morever, we detected that neuronal apoptosis marker active caspase-3 had co-localizations with Vps4A. Additionaly, Vps4A knockdown in vitro specifically leads to decreasing neuronal apoptosis coupled with increased Akt phosphorylation. All datas suggested that Vps4A was involved in promoting neuronal apoptosis via inhibiting Akt phosphorylation after ICH.