Dehydration and Estrous Staging in the Rat Larynx: an in vivo Prospective Investigation, Publication date: Available online 13 July 2019 Source: Journal of Voice Author(s): Abigail Cox, Naila Cannes do Nascimento, Andrea Pires dos Santos, M. Preeti Sivasankar Abstract 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.