Water Versus Electrolyte Rehydration on Vocal Fold Osmotic and Oxidative Stress Gene Expression.

OBJECTIVES: Systemic dehydration may induce osmotic and oxidative stress in the vocal folds, but our knowledge of the biology and mitigation with rehydration is limited. The purpose of this experiment was to evaluate whether systemic dehydration induces vocal fold oxidative and osmotic stress and to compare the impact of rehydration by water intake versus electrolyte intake on osmotic and oxidative stress-related gene expression. METHODS: Four-month-old male Sprague-Dawley rats (N = 32) underwent water restriction. Rehydration was achieved with ad libitum access to water or electrolytes for 24 hours. Rats were divided into four groups: euhydration control, dehydration-only, dehydration followed by either water or electrolyte rehydration (n = 8/group). Gene expression was assessed via RT2 Gene Expression Profiler arrays. RESULTS: With respect to oxidative stress, 10 genes were upregulated and 2 were downregulated after vocal fold dehydration compared with the euhydrated control. Concerning osmotic stress, six genes were upregulated with dehydration only, six genes were upregulated following rehydration with water, whereas a single gene was upregulated with electrolyte rehydration. All genes with significantly different expression between the rehydration groups showed lower expression with electrolytes compared with water. CONCLUSIONS: The results support a potential role of oxidative and osmotic stresses in vocal folds related to systemic dehydration. The differences in stress-related gene expression in vocal fold tissue between rehydration with electrolytes or water, albeit modest, suggest that both rehydration options offer clinical utility to subjects experiencing vocal fold dehydration with preliminary evidence that electrolytes may be more effective than water in resolving osmotic stress. LEVELS OF EVIDENCE: NA (prospective animal study) Laryngoscope, 2024.

Temporal gene expression during asexual development of the apicomplexan Sarcocystis neurona.

Asexual replication in the apicomplexan Sarcocystis neurona involves two main developmental stages: the motile extracellular merozoite and the sessile intracellular schizont. Merozoites invade host cells and transform into schizonts that undergo replication via endopolygeny to form multiple (64) daughter merozoites that are invasive to new host cells. Given that the capabilities of the merozoite vary significantly from the schizont, the patterns of transcript levels throughout the asexual lifecycle were determined and compared in this study. RNA-Seq data were generated from extracellular merozoites and four intracellular schizont development time points. Of the 6,938 genes annotated in the S. neurona genome, 6,784 were identified in the transcriptome. Of these, 4,111 genes exhibited significant differential expression between the merozoite and at least one schizont development time point. Transcript levels were significantly higher for 2,338 genes in the merozoite and 1,773 genes in the schizont stages. Included in this list were genes encoding the secretory pathogenesis determinants (SPDs), which encompass the surface antigen and SAG-related sequence (SAG/SRS) and the secretory organelle proteins of the invasive zoite stage (micronemes, rhoptries, and dense granules). As anticipated, many of the S. neurona SPD gene transcripts were abundant in merozoites. However, several SPD transcripts were elevated in intracellular schizonts, suggesting roles unrelated to host cell invasion and the initial establishment of the intracellular niche. The hypothetical genes that are potentially unique to the genus Sarcocystis are of particular interest. Their conserved expression patterns are instructive for future investigations into the possible functions of these putative Sarcocystis-unique genes. IMPORTANCE: The genus Sarcocystis is an expansive clade within the Apicomplexa, with the species S. neurona being an important cause of neurological disease in horses. Research to decipher the biology of S. neurona and its host-pathogen interactions can be enhanced by gene expression data. This study has identified conserved apicomplexan orthologs in S. neurona, putative Sarcocystis-unique genes, and gene transcripts abundant in the merozoite and schizont stages. Importantly, we have identified distinct clusters of genes with transcript levels peaking during different intracellular schizont development time points, reflecting active gene expression changes across endopolygeny. Each cluster also has subsets of transcripts with unknown functions, and investigation of these seemingly Sarcocystis-unique transcripts will provide insights into the interesting biology of this parasite genus.

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.

Dehydration and Estrous Staging in the Rat Larynx: an in vivo Prospective Investigation.

OBJECTIVE: This novel study sought to untangle the association between hydration state and the estrous cycle in the vocal folds, since the voice is reported to negatively change in speakers during the estrous cycle and with dehydration. We hypothesized that there would be alterations in vocal fold tissue morphology depending on hydration state and that these changes would vary with the estrous cycle. STUDY DESIGN: Prospective, in vivo study design. METHODS: Female Sprague Dawley rats (n = 30) were used in this study. Sixteen rats were systemically dehydrated to an average of 10% reduction in body weight by withholding water (range of body weight loss: 8%-13%). Fourteen rats were assigned to euhydrated, control condition. Estrous stage of female Sprague Dawley rats (n = 30) was determined via cytological evaluation of vaginal smears. Following euthanization, larynges were prepared for histological staining with hematoxylin and eosin, Masson's trichrome and alcian blue (pH 2.5). To quantify hyaluronan, alcian blue staining was completed pre- and posthyaluronidase incubation. The change in staining percent was quantified with image analysis algorithms and reported as the hyaluronan quantity. Relative collagen distribution (index of dehydration), hyaluronan quantity, and tissue morphology were the outcome measures. RESULTS: Systemic dehydration was associated with changes in hyaluronan quantity in the rat vocal fold lamina propria. Dehydration did not significantly affect the collagen distribution nor the tissue morphology. Estrous stage alone does not impact the quantity of vocal fold hyaluronan, alter tissue morphology, or change collagen distribution. CONCLUSION: Decreases in hyaluronan quantity in the lamina propria of the rat vocal fold may play a role in tissue fluid balance during systemic dehydration. Future studies will expand this work to investigate additional components of the vocal fold extracellular matrix to fully elucidate the impact of hydration state on the vocal fold.

Unraveling the molecular pathobiology of vocal fold systemic dehydration using an in vivo rabbit model.

Vocal folds are a viscoelastic multilayered structure responsible for voice production. Vocal fold epithelial damage may weaken the protection of deeper layers of lamina propria and thyroarytenoid muscle and impair voice production. Systemic dehydration can adversely affect vocal function by creating suboptimal biomechanical conditions for vocal fold vibration. However, the molecular pathobiology of systemically dehydrated vocal folds is poorly understood. We used an in vivo rabbit model to investigate the complete gene expression profile of systemically dehydrated vocal folds. The RNA-Seq based transcriptome revealed 203 differentially expressed (DE) vocal fold genes due to systemic dehydration. Interestingly, function enrichment analysis showed downregulation of genes involved in cell adhesion, cell junction, inflammation, and upregulation of genes involved in cell proliferation. RT-qPCR validation was performed for a subset of DE genes and confirmed the downregulation of DSG1, CDH3, NECTIN1, SDC1, S100A9, SPINK5, ECM1, IL1A, and IL36A genes. In addition, the upregulation of the transcription factor NR4A3 gene involved in epithelial cell proliferation was validated. Taken together, these results suggest an alteration of the vocal fold epithelial barrier independent of inflammation, which could indicate a disruption and remodeling of the epithelial barrier integrity. This transcriptome provides a first global picture of the molecular changes in vocal fold tissue in response to systemic dehydration. The alterations observed at the transcriptional level help to understand the pathobiology of dehydration in voice function and highlight the benefits of hydration in voice therapy.