Sex hormones are necessary to enable respiratory neuroplasticity, including phrenic long-term facilitation (pLTF), a form of respiratory motor plasticity elicited by acute, intermittent hypoxia (AIH). Female rats exhibit a progressive increase in phrenic nerve amplitude after AIH characteristic of pLTF only during pro-oestrus, the stage of the oestrous cycle notable for elevated circulating oestradiol levels. Removal of the ovaries [ovariectomy (OVX)], the primary source of circulating oestradiol, also eliminates AIH-induced pLTF after 1 week. Ovariectomy is used routinely as a model to examine the impact of sex hormones on CNS structure and function, but the long-term impact of OVX is rarely examined. Extra-ovarian sites of oestradiol synthesis, including multiple CNS sites, have been identified and might possess the capacity to restore oestradiol levels, in part, over time, impacting respiratory function and the expression of respiratory neuroplasticity. We examined both ventilation in awake, freely behaving female rats, using barometric plethysmography, and the expression of AIH-induced pLTF in anaesthetized, ventilated female rats 2 and 12 weeks after OVX and compared them with age-matched ovarian-intact female rats. Our findings indicate that chronic OVX had little impact on baseline breathing or in the response to respiratory challenge (10% O 2 , 5% CO 2 , balance N 2 ) during plethysmography. However, OVX rats at both 2 and 12 weeks demonstrated a persistent loss of AIH-induced pLTF relative to control animals (P < 0.01), suggesting that other sources of oestradiol synthesis were insufficient to restore pLTF. These data are consistent with our previous work indicating that oestradiol plays a key role in expression of AIH-induced respiratory neuroplasticity.

Female carriers of Duchenne muscular dystrophy (DMD) presenting with DMD symptomology similar to males with DMD, such as skeletal muscle weakness and cardiomyopathy, are termed manifesting carriers. There is phenotypic variability among manifesting carriers including the age of onset, which can range from the first to fourth decade of life. In females, estrogen levels typically begin to decline during the fourth decade of life and estrogen deficiency contributes to loss of muscle strength and recovery of strength following injury. Thus, we questioned whether the decline of estrogen impacts the development of DMD symptoms in females. To address this question, we studied 6–8 month-old homozygous mdx female mice randomly assigned to a sham or ovariectomy (OVX) surgical group. In vivo whole-body plethysmography assessed ventilatory function and diaphragm muscle strength was measured in vitro before and after fatigue. Anterior crural muscles were analyzed in vivo for contractile function, fatigue, and in response to eccentric contraction (ECC)-induced injury. For the latter, 50 maximal ECCs were performed by the anterior crural muscles to induce injury. Body mass, uterine mass, hypoxia-hypercapnia ventilatory response, and fatigue index were analyzed by a pooled unpaired t-test. A two-way ANOVA was used to analyze ventilatory measurements. Fatigue and ECC-injury recovery experiments were analyzed by a two-way repeated-measures ANOVA. Results show no differences between sham and OVX mdx mice in ventilatory function, strength, or recovery of strength after fatigue in the diaphragm muscle or anterior crural muscles (p ≥ 0.078). However, OVX mice had significantly greater eccentric torque loss and blunted recovery of strength after ECC-induced injury compared to sham mice (p ≤ 0.019). Although the results show that loss of estrogen has minimal impact on skeletal muscle contractile function in female mdx mice, a key finding suggests that estrogen is important in muscle recovery in female mdx mice after injury.

Sex hormones significantly influence respiratory function and the expression of respiratory neuroplasticity. For example, 17ß‐ estradiol (E2), the most prevalent and neuroactive sex steroid in females, is necessary for expression of phrenic long‐term facilitation (pLTF), a well characterized form of plasticity induced by acute intermittent hypoxia (AIH) in both females and males. To meet this requirement for E2, males aromatize testosterone to E2 in order to express respiratory neuroplasticity. However, on its own (i.e., without aromatase conversion to E2), testosterone has also been shown to enhance breathing. Since both E2 and testosterone have individually demonstrated the capacity to enhance respiratory function, we hypothesized that supplementation of these sex hormones would improve respiratory function in rats 2 weeks post experimental cervical spinal cord injury (SCI). Adult male Sprague‐Dawely rats received a C2 hemi‐section SCI (or Sham laminectomy) and recovered with standard post‐op care. One‐week post‐injury, a controlled‐release hormone pellet was placed subcutaneously in SCI rats to deliver a pre‐determined dose of daily hormone supplementation for the duration of the study. Pellets contained either E2 (β‐estradiol 3‐benzoate, 10 μg/day), 5α‐dihydrotestosterone (DHT), a testosterone derivative that is not converted to E2 (50 μg/day), or blank placebo pellets (50 μg/day). Respiratory function was measured 7 days later (2 weeks post SCI) using Whole‐Body Barometric Plethysmography. Consistent with prior reports, our preliminary findings demonstrated that placebo treated SCI rats breathed with increased respiratory frequency, but reduced tidal volume production. DHT supplementation had little impact on these respiratory parameters, with values remaining similar to placebo control rats. However, one week of E2 supplementation led to reductions in respiratory frequency and increased tidal volume production during baseline room‐air conditions and in response to a strong respiratory challenge (10.5% O2/5% CO2), suggesting improved overall ventilation. These data suggest that estrogen supplementation may facilitate improvements in breathing function in the early stages of spinal cord injury recovery. Support or Funding Information This work was supported by funding from The Minnesota Office of Higher Education, The University of Minnesota Division of Physical Therapy, and The University of Minnesota Department of Rehabilitation Medicine

Estrogen impacts neuroplasticity in multiple areas of the central nervous system including within respiratory neural networks. Previous research from our group established that estrogen is necessary to permit expression of phrenic long‐term facilitation (pLTF), a form of respiratory neuroplasticity induced by acute, intermittent hypoxia (AIH). Many factors influence circulating estrogen levels such as age, gender, and diet. Specifically, the Western Diet is associated with increased levels of circulating estrogen in post‐menopausal women. Common to cultures of the Western Hemisphere, the Western Diet is characterized by high intakes of red and processed meats, sweets, fried foods, and refined grains with the addition of a high intakes of sugar, salt, omega 6 fatty acids, and a reduction of omega 3 fatty acids. Since the Western Diet is associated with high estrogen levels, and estrogen appears to play a critical role in expression of respiratory neuroplasticity, we assessed whether rats fed a Western Diet would have altered respiratory function and expression of AIH‐induced pLTF. Experimental rats of both sexes were fed a Western Diet in pellet form for 12 weeks and compared to rats consuming standard pellet chow. Weekly whole body plethysmography was used to measure ventilation and changes in ventilatory responses over time. At the conclusion of 12 weeks, AIH‐induced phrenic LTF was measured in anesthetized, vagotomized, and mechanically ventilated rats. Surprisingly, neither sex exhibited a change in body weight in response to the 12‐wk Western Diet, nor was baseline ventilation different between experimental groups within the same sex. However, with hypoxic challenge, male rats on the Western Diet showed lower breathing frequency and higher tidal volumes, while females on the Western Diet exhibited the opposite effects. Also, AIH‐induced pLTF was abolished in male and female Western Diet rats despite an increase in circulating estrogen levels in Western Diet Rats as measured by ELISA assay. These preliminary data suggest that the Western Diet may induce progressive changes in ventilation and loss of respiratory neuroplasticity independent of increased circulating estrogen levels.

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 ...