Ovariectomy at 3 or 10 weeks of age does not affect the ventilatory response to hypoxia in adult rats ()
1. INTRODUCTION
In adult mammals, including humans, an important body of evidence shows that the ovarian hormones progesterone and estradiol are potent respiratory stimulants [1,2]. Both hormones act on peripheral (via carotid body chemoreceptors) and central respiratory control systems (via the medullary nucleus) to increase breathing in response to either hypoxia or hypercapnia [2,3] . Indeed, these hormones are considered to have a protective role against disrupted breathing during sleep in females before the menopausal period [1,2,4] . Ovariectomy is a standard approach used to study the effect of ovarian hormones on the hypoxic ventilatory response. However, the results obtained with this approach have often been contradictory [1,5] . That contradiction may partly depend on the delay between the surgery modulating the level of circulating ovarian hormones and the assessments of ventilation. In this regard, we tested the hypothesis that performing ovariectomy at an early age and not at an adult age disrupts the hypoxic response in rats. Ventilation at rest and in response to hypoxia was measured in adult female rats (12 weeks old) that were ovariectomized either at 3 (young rats, prepubertal) or 10 weeks old (adult). Concomitantly, the plasma levels of ovarian hormones were assessed to evaluate the potential relationship between hormone levels and ventilation.
2. MATERIALS AND METHODS
2.1. Animals and Ventilatory Recording
Experiments were performed on 12-week-old female Sprague-Dawley rats (n = 41) born and raised in our animal care facilities. The animals originated from 6 different litters to reduce litter-specific effects. Rats were supplied with food and water ad libitum and maintained under standard animal care conditions (21˚C, 12 h:12 h dark:light cycle, lights on at 8:00 A.M. and off at 8:00 P.M.). Mating, litter manipulation, surveillance and ventilatory recording using whole body, flow-through plethysmography were all performed according to our standard procedures, which have been previously described in detail [6,7] . Ventilatory parameters were first recorded under baseline ventilation (FiO2 = 21%) for 10 min, and rats were then exposed to moderate hypoxia (FiO2 = 12%) for 20 min. On the day of the experiment, each rat was acclimated to the plethysmographic chamber (model PLY 3223, Buxco Electronics, Sharon, CT) for approximately 30 min before the baseline recording. Body temperature was measured rectally at the end of the baseline recording and hypoxic exposure using a thermocouple for rodents (Harvard, Holliston, MA, USA). Baseline measurements were made when the animal was quiet but awake and ventilatory activity was stable. Breathing frequency (fr/ min) and tidal volume (Vt ml BTPS/100 g) were measured using data acquisition software (IOX, EMKA Technologies, Falls Church, VA, USA) and were used to calculateminute ventilation (VE = fr × VT ml/min/100g). Barometric pressure, chamber temperature, and humidity were measured to correct VT as previously described ADDIN EN.CITE ADDIN EN.CITE.DATA [6, 7] . The flow of gas into the plethysmography chamber was maintained constant at 2.0 L/min. The oxygen concentration flowing in and out of the chamber was continuously measured (Oxygen analyzer model S-3A, Ametek, Pittsburgh, PA, USA) and used for subsequent calculations of oxygen consumption (VO2 = ml/min/100 g) ADDIN EN.CITE ADDIN EN.CITE.DATA [6,7] . Oxygen consumption was assessed at baseline and at the end of the hypoxic exposure. All experiments were performed between 9:00 and 12:00 A.M.
All protocols were in accordance with the Canadian Council on Animal Care Guidelines and were approved by the Laval University Animal Protection Committee.
2.2. Surgery
Rats underwent sham surgery (intact) or ovariectomy (OVX) under isoflurane anesthesia (2% - 2.5% in air) at 3 (young rats, prepubertal) or 10 weeks (adult) of age. Briefly, a small midline abdominal incision was made, the adipose tissue containing the ovaries was gently pulled, the ducts and blood vessels connected to the ovaries were sutured and then cut, and the ovaries were removed. Shamoperated rats underwent an identical intervention but without blood vessel ligation or ablation of the ovaries. Each rat received standard post-surgical care, which included buprenorphine (5 mg/kg, subcutaneously), 5 ml of Ringer’s lactate (subcutaneously; only for adult rats) and an antibiotic (Baytril: 5 mg/kg/d, for a total of 3 d). In all rats, the hypoxic response was then evaluated at 12 weeks of age, independent of surgery time.
2.3. Plasma Hormones
To exclude any possible effects of hypoxia on steroid levels, progesterone and estradiol were assessed in rats (5 - 6 in each group) issued from 3 other litters not used for the hypoxic response experiments described above. Rats were deeply anesthetized with isoflurane (~5%), and blood was taken by cardiac puncture.The samples were centrifuged immediately and stored at –20˚C until analysis. Progesterone and estradiol levels were assessed using standard chemiluminescence kits (Roche Diagnostics, Laval, QC, Canada) as previously described [7]. All ventilatory measurements and plasma hormone samplings were performed between 9:00 and 12:00 A.M.
2.4. Data Analyses and Statistic
Respiratory frequency, tidal volume, and minute ventilation were first calculated on a minute-by-minute basis using data analysis software (Data analyst, EMKA Technologies). The baseline value represents the mean of 5 consecutive min of steady respiration before exposure to hypoxia. The temporal dynamics of the ventilatory variables in response to hypoxia were averaged every 2 min over the first 10 min of exposure (early phase), while the last 10 min were averaged in 5-min epochs (late phase) and expressed as the percent (%) change from baseline. An ANOVA for one or multiple factors, including the age at the time of surgery (young vs. adult), type of surgery (sham vs. OVX), and condition (hypoxia), was used (Stat View 4.5). A p < 0.05 was considered statistically significant. Data are expressed as the mean ± SEM.
3. RESULTS
3.1. Ovariectomy Does Not Affect the Baseline Variables
Table 1 shows the baseline variables measured in sham (intact) or ovariectomized (OVX) rats. Body weight and body temperature were comparable in all groups independent of their age when surgery was performed (i.e., 3 or 10 weeks old) (Table 1). The baseline ventilatory variables and oxygen consumption were also similar between all groups of rats (Table 1).