Objective
Obstructive sleep apnea, a breathing disorder caused by the repetitive collapse of the upper airway during sleep, results in a state of chronic intermittent hypoxia (CIH). Although the etiology and consequences of CIH are extensively investigated in the adult, the developmental ramifications of this disease process are unknown.
Design
This study was done to investigate the effect of CIH during gestation on offspring development. Pregnant female Spraque-Dawley rats were exposed to daily CIH throughout the gestational period.
Results
Postnatal day-1 offspring from CIH mothers were asymmetrically growth restricted, with decreased body weights and elevated brain-weight:liver-weight ratios. Furthermore, CIH newborns had elevated heart- and brain-weight:body weight ratios, and decreased liver-weight:body weight ratios. By adulthood, body weights of growth restricted offspring were significantly greater, as were the liver-weight:body weight ratios. CIH offspring also had greater body fat deposition, were hyperglycemic and had elevated plasma levels of insulin during development into adults.
Conclusion
These data suggest that alteration of the maternal intrauterine environment by gestational CIH effects the long-term development of the offspring and increases the risk of the offspring to metabolic diseases in adulthood.
Obstructive sleep apnoea (OSA) is a breathing disorder caused by upper airway collapse during sleep, resulting in an intermittent reduction or complete blockage of airflow. Repetitive OSA occurrences result in chronic intermittent hypoxia (CIH), characterized by frequent decreases in blood O 2 saturation. This hypoxic condition is corrected and breathing resumes following a surge in autonomic activity that awakens the sleeping individual and reopens the airway. OSA is associated with a variety of comorbidities, including chronic hypertension, obesity, and insulin resistance. Although recent efforts have placed importance on understanding the consequences of OSA and the underlying state of CIH, the cross-generational effects of this condition have not been investigated.
Data exist suggesting that developmental deficits during gestation increase the risk of morbidity and mortality from cardiovascular and metabolic diseases in adult life. These deficits are thought to result from intrauterine growth restriction (IUGR), where the fetus does not reach its growth potential. Although there are a variety of complications that can result in IUGR, the most common occurrence involves a decrease in the supply of O 2 to the fetus, that is vital for normal growth and development, which can be triggered by placental insufficiency, smoking, pulmonary diseases, and maternal hypoxic distress. A chronic hypoxic stress during gestation creates a maternal intrauterine environment that does not adequately provide O 2 and nutrients to the fetus. Thus, growth is restricted in an effort to create a balance between the decreased supply and consumption of O 2 . The low birthweight caused by IUGR represents a common pathologic condition diagnosed during pregnancy.
There is a well-described link between developmental deficits and increased incidence of perinatal mortality. The Barker hypothesis, highlighting the importance of prenatal development puts forth the idea that an inadequate supply of oxygen and nutrients creates an intrauterine environment that causes structural and functional adaptations during organogenesis. It is these changes that are believed to increase the susceptibility of an individual to cardiovascular and metabolic diseases later in life.
This study was done to examine the immediate and long-term changes in offspring development caused by suboptimal growth conditions resulting from CIH. This was done using a rodent model for investigating the effects of CIH exposure on pregnancy and offspring development which mimics the clinical changes in O 2 availability experienced by patients with OSA.
Materials and Methods
Adult female and male Sprague-Dawley rats (250 g; n = 14) were obtained from Charles River Canada (Saint-Constant, Quebec, Canada). All animals were housed individually in standard cages in rooms maintained at constant temperature (23°C) and humidity (65%) with a 12 hours light/dark cycle beginning at 0700 hours. Food and water were available to all animals ad libitum. All experimental procedures were performed in accordance with the Guide to the Care and Use of Experimental Animals by the Canadian Council on Animal Care and approved by the Animal Care Committee at the University of Western Ontario.
All animals were allowed 1-week acclimatization period following arrival during which the reproductive cycle of each female rat was followed. The stage of the oestrous cycle was confirmed daily through vaginal smear. On confirmation of the proestrous stage, the females were mated with 1 male for each female. Successful impregnation was confirmed by the presence of sperm in a vaginal smear taken the following morning.
Chronic intermittent hypoxia
The confirmation of pregnancy marked gestational day-0, at which point females were separated from males and housed individually. The following morning (gestational day-1), pregnant females (n = 8) were exposed daily to CIH inside a hypoxia chamber with built-in plexiglass tubes (10 cm diameter by 35 cm length) for housing individual rats, and each rat was exposed to alternating cycles of normoxic (room air; 120 seconds) and hypoxic (6.5% O 2 nadir; 80 seconds) conditions at a frequency of 18 cycles/hour. Conditions within the chamber were isobaric (770 ± 11 mm Hg) and eucapnic (< 0.1% CO 2 ). Control animals (n = 6) were placed in an adjacent chamber that continuously cycled room air. The duration of exposure for both groups was 8 hours (1000 hours to 1800 hours) per day from gestational day-1 to day-20. Body weights of all rats were recorded prior and immediately following daily CIH exposure.
Measurement of food and water intake
All females and offspring were allowed standard rat chow for the duration of the study. Food and water intake measurements for each animal were obtained between 1800 hours and 1000 hours the next day and values were standardized to the daily pre-exposure body weight of each rat.
Postdelivery
After the final exposure to CIH, females were returned to the animal facility for an undisturbed delivery. Immediately following birth (postnatal day-1), offspring from each mother were counted and sexed within each litter using anogenital distance. In all cases, the litter size was then reduced to 4 males. All additional males from each litter were sacrificed for analysis of neonatal offspring development. The 4 males were housed with the mother until postnatal day-21, at which point the pups were weaned and separated into pairs. All offspring had standard rat chow and water available ad libitum until adulthood (12-weeks of age).
Offspring tissue collection
Postnatal day-1 offspring were weighed and then sacrificed via decapitation. An incision was made along the midline to expose the thoracic and abdominal cavities to remove heart and liver that were immediately weighed and snap frozen in liquid nitrogen. An incision was made on the dorsal surface of the head to remove the skull and extract the brain that was immediately weighed and frozen on dry ice.
Four males from each litter were allowed to reach adulthood. From these offspring, 2 were sacrificed at 6-weeks of age and the other 2 at 12-weeks of age. All animals were fasted overnight before sacrifice. Under equithesin anesthesia (3 mL/kg, ip), blood samples from each rat were collected via intracardiac puncture, and then the heart and liver were removed, weighed, and snap frozen in liquid nitrogen. Brains were extracted, weighed, and frozen on dry ice. Epididymal and retroperitoneal fat pads were removed bilaterally and weighed.
Blood glucose and insulin measurement
Blood collected from 6- and 12-week old males was aliquoted into 1 mL centrifuge tubes containing 10 μL of 7% ethylenediaminetetraacetic acid (EDTA) dissolved in water. The samples were placed in a refrigerated centrifuge and spun at 10,500 g for 15 minutes at 4°C. The plasma layer was aspirated and stored at −80°C until assayed.
Fasting blood glucose was measured using a standard glucometer. Values were obtained at the time of sacrifice by sampling blood extracted from the heart via intracardiac puncture. Plasma insulin measurements were made using a rat-specific enzyme immunoassay for insulin (Cat.80 INSRT-E01; ALPCO Diagnostics, Salem, NH). Absorbance readings of insulin assays were determined using a Spectramax M5 plate reader (Molecular Devices, Downingtown, PA).
Assay sensitivity was calculated as the mean plus 2 standard deviations for the zero standards in the insulin enzymatic immunoassay and determined to be 0.130 ng/mL. Intraassay variability was calculated as the mean of the coefficients of variation for all samples measured in the assay. The intraassay variability of the insulin immunoassay was 5.2%.
Intraperitoneal glucose tolerance test
Glucose tolerance was measured in 6- and 12-week old male offspring. Rats were fasted overnight before commencement of the test. Following a baseline measurement of blood glucose levels, rats were administered an injection of D-glucose (40% solution; 2 g/kg body weight, ip). Blood glucose measurements were taken at 5, 10, 15, 30, 60, and 120 minutes postinjection using a standard glucometer by sampling tail vein blood.
Data and statistical analysis
All data are expressed as mean ± standard deviation. Maternal body weight and consumption data were analyzed using a 2-way analysis of variance. Organ weights were measured in grams and were expressed as a ratio standardized to body weight. All offspring results, glucose and insulin concentrations were compared using a 2-tailed Student t test to determine statistical significance. Glucose tolerance test data were analyzed using a 2-way analysis of variance. In all comparisons, a minimum P value of < .05 was taken to indicate statistical significance (GraphPad Prism; GraphPad Software, San Diego, CA).
Results
Effect of CIH on pregnant female
During the first week pregnant female subjected to CIH treatment had no changes in body weight ( Figure 1 , A). However, from gestational day-8 to day-20, CIH mothers showed a daily increase in body weight ( Figure 1 , A). On the other hand, mothers exposed to normoxia during pregnancy showed increases in body weight from gestational day-1 to day-20 ( Figure 1 , A). The body weight of normoxic control mothers was significantly greater than that of CIH mothers from gestational day-11 to day-20. Furthermore, the cumulative weight gained by normoxic mothers during gestation was significantly greater than that of CIH mothers from gestational day-6 to day-20 ( Figure 1 , B).
No differences in total daily caloric intake were observed throughout gestation between mothers exposed to CIH or normoxia ( Figure 1 , C). However, mothers exposed to CIH during the gestational period had significantly greater water intake compared with normoxic mothers ( Figure 1 , D).
Mothers subjected to 8 hours of CIH experienced significantly greater daily weight loss during exposure ( Figure 2 ). In addition, weight loss data collected from control mothers was compared with a subset of pregnant females that were kept in their home cages and not placed within the chambers, but had no access to food and water for the 8-hour period. The weight loss experienced by mothers in the normoxic chamber was not different from that of mothers kept in their home cages (data not shown).
Effect of CIH on offspring litter size, body weight and organ weights
There was no difference in the number of offspring in the litters of CIH and normoxic mothers ( Figure 3 , A). Furthermore, there was no difference in the number of male or female offspring produced in litters of either group. The litters of both CIH and normoxic mothers had a significantly greater proportion of female offspring in comparison to the number of male offspring (CIH, 9 ± 1 females vs 6 ± 1 males; Normoxia, 8 ± 1 females vs 5 ± 1 males).
Weight measurements taken on postnatal day-1 show that offspring from mothers exposed to CIH during gestation had significantly lower body weights compared with offspring from normoxic mothers ( Figure 3 , B). Brain weight was standardized to the liver weight within each offspring, a classical measurement for characterizing asymmetric growth restriction. CIH offspring were found to have significantly greater brain-weight:liver-weight ratios ( Figure 3 , C). The organ weights of the heart and brain were not different between the offspring of CIH and normoxic exposed mothers. The livers of CIH offspring weighed significantly less than the livers of normoxic offspring. When the organ weights of each offspring were standardized to its body weight, pups from mothers exposed to CIH had significantly greater heart-weight:body-weight and brain-weight:body-weight ratios ( Figure 3 , D and E, respectively). Furthermore, these offspring had lower liver-weight:body-weight ratio ( Figure 3 , F).
At 6 weeks of age, body weights of CIH offspring remained significantly lower ( Figure 4 , A). Standardized heart and brain weights were not different between the offspring of the CIH and normoxic mothers ( Figure 4 , B and C, respectively). Furthermore, there was no difference in the liver-weight:body-weight ratios between the 2 groups ( Figure 4 . D). However, CIH offspring had significantly greater epididymal and retroperitoneal fat mass compared with offspring of the normoxic mothers ( Figure 4 , E and F, respectively).
By 12 week of age, offspring of the CIH mothers had outgrown the normoxic offspring and had a higher mean body weight ( Figure 5 , A). Standardized heart and brain weights were not different between CIH and normoxic offspring ( Figure 5 , B and C, respectively). Offspring of CIH exposed mothers had significantly greater liver-weight:body-weight ratios ( Figure 5 , D). In addition, CIH group offspring had significantly greater epididymal and retroperitoneal fat mass compared with normoxic offspring ( Figure 5 , E and F, respectively).
