The Long‐Term Impact of Ovariectomy on Ventilation and Expression of Phrenic Long‐Term Facilitation in Female Rats

Abstract

Estrogen is necessary for the expression of phrenic long-term facilitation (pLTF) in female rats. pLTF, a well-studied model of respiratory neuroplasticity, is characterized by a progressive increase in phrenic nerve amplitude following acute intermittent hypoxia (AIH). Gonadally-intact, adult female rats only develop pLTF in stages of the estrous cycle with high levels of circulating estrogen. Removal of the ovaries (ovariectomy, OVX), the primary source of circulating estrogen in females, also prevents AIH-induced pLTF and estrogen supplementation is sufficient to restore pLTF in OVX rats, supporting a key role for estrogen in the development of pLTF. Respiratory neuroplasticity studies using OVX female rats are generally completed ~7-10 days post-OVX; a timeframe sufficient for surgery recovery and for circulating estrogen levels to significantly decline. However, few studies have explored the long-term impact of OVX on respiratory function or the development of respiratory neuroplasticity. Here, we examined ventilation in awake, freely behaving rats and the development of AIH-induced pLTF in adult female rats 12 weeks-post OVX (or sham control surgeries) and compared them to female rats 2 weeks-post OVX. Our hypothesis was that chronic OVX would have minimal long-term impact on ventilation but would continue to impair development of AIH-induced pLTF. Whole body plethysmography was used to measure baseline breathing (20.9% O2, N2 balance) and ventilatory responses to strong chemoreceptor stimulation (10% O2, 5% CO2, N2 balance). Respiratory measures including frequency, tidal volume, minute ventilation, and respiratory neural drive were measured and compared between groups. Following plethysmography, phrenic nerve recordings were used to quantify pLTF. Results indicate that chronic OVX had little impact on baseline breathing function and rats in all experimental groups responded appropriately to respiratory challenge during plethysmography. However, 12 week OVX rats showed a significant increase in tidal volume relative to baseline during respiratory challenge (p≤0.001), different from other groups. This increase in tidal volume production was also reflected in the calculation of tidal volume/inspiratory time, a measure of relative respiratory neural drive (p≤0.01) but not in minute ventilation. OVX had sustained effects on development of respiratory plasticity, as only rats in the 12 week sham control group displayed pLTF, indicated by significantly increased phrenic nerve amplitude 60-min following AIH (p≤0.001). This increase in amplitude was higher than both 12 week and 2 week OVX groups (p≤0.01). ELISA assays also showed a significant correlation between circulating estrogen levels and magnitude of pLTF (p= 0.039) In summary, OVX appears to limit the expression of respiratory neuroplasticity in female rats for up to 12 weeks. Since no significant changes in circulating estrogen are observed in OVX rats over that time, these data are consistent with our prior work indicating that estrogen plays a key role in development of respiratory neuroplasticity.