Neonatal stress increases respiratory instability in rat pups
Introduction
In the premature infant, respiratory instability and apneas are a major cause of hospitalisation and morbidity generally attributed to the immaturity of the chemoreceptors (both central and peripheral) and of the neural circuits that generate respiratory motor outflow (Abu-Shaweesh and Martin, 2008, Cardot et al., 2007, Gauda et al., 2004, Gaultier and Gallego, 2005, Nock et al., 2004). The proximal mechanisms responsible for respiratory control dysfunction in premature newborn are not fully understood but in newborn rat, exposure to intermittent hypoxia (a consequence of recurrent apneas) leads to chemoreceptor hypersensitivity (Peng et al., 2004). Since this condition, in addition to an inherently unstable respiratory rhythm, contributes to periodic breathing and apnea in newborn (Al-Matary et al., 2004, Gauda et al., 2007, Gaultier and Gallego, 2008, Greer et al., 2006, Khoo, 2000), intermittent hypoxia associated with repeated apneas may exacerbate respiratory control disorder in premature newborn.
During early life, repeated stimulation of peripheral chemoreceptors influences the developmental trajectory of the respiratory control system to produce diverse physiological phenotypes (Bavis and Mitchell, 2008, Carroll, 2003, Cayetanot et al., 2009, Kinkead and Gulemetova, 2010). While hypoxia per se likely has a direct effect on respiratory control development, we cannot ignore the fact that hypoxia also stimulates important neuroendocrine responses such as ACTH and glucocorticoid release (Jacobson and Dallman, 1989, Raff and Roarty, 1988). Moreover, intermittent hypoxia sensitizes acute neuroendocrine responsiveness to stress (Ma et al., 2008). These data, combined with the detrimental effects of early life exposure to stress on brain maturation (Field and Diego, 2008, Matthews, 2001, Van den Hove et al., 2006), raise the possibility that stress hormones contribute to abnormal respiratory control in the newborn.
Research on humans and animal models show that insufficient or inadequate mother–infant interactions during early life is stressful to the newborn and has significant and persistent consequences on memory, behaviour, and cognition (Gunnar, 2003, Hsu et al., 2003, Kuhn and Schanberg, 1998, Levine, 2005, Renard et al., 2007, Slotten et al., 2006). In rat, neonatal maternal separation (NMS) also interferes with the development of primary homeostatic functions since NMS has persistent and sex-specific effects on respiratory control development (Kinkead and Gulemetova, 2010). Although NMS (3 h/day from P3 to P12) poses no direct threat to respiratory homeostasis, adult male (but not female) rats previously subjected to NMS are hypertensive and show an augmented hypoxic ventilatory response (Genest et al., 2007a, Genest et al., 2004, Kinkead et al., 2008, Kinkead and Gulemetova, 2010), the latter effect is correlated with a greater respiratory variability during non-REM sleep (Kinkead et al., 2009). While we are beginning to appreciate the mechanisms and potential clinical significance of this intriguing manifestation of developmental plasticity (Kinkead and Gulemetova, 2010), we still do not know whether the effects of NMS on respiratory regulation are apparent in young (pre-pubertal) rats.
Based on previous observations in adult rats, we tested the hypothesis that NMS augments respiratory instability and O2 chemoreflex in 12 days old animals. To do so, we measured respiratory activity in rat pups in the hours that follow cessation of the NMS protocol using whole body plethysmography. We quantified various indicators of respiratory instability (coefficient of breathing cycle variation, apnea index) under “resting” (normoxic) conditions; the O2 chemoreflex was then assessed by measuring the increase in breathing frequency that takes place at the onset of hypoxia. Data were compared between P12 pups previously subjected to our standard NMS protocol and undisturbed pups (control). Experiments were performed on male and female rats to assess sexual dimorphism.
Section snippets
Materials and methods
Experiments were performed on 33 NMS and 34 control (undisturbed) 12 days old male and female Sprague–Dawley rats (see Table 1 and Fig. 1 for details of animal repartition amongst groups). Male and female genitors were purchased (Charles River Canada, St. Constant, QC, Canada), but all animals used in this study were born and raised in our animal care facilities. Rats were supplied with food and water ad libitum and maintained in standard laboratory conditions (21 °C, 12:12-h dark–light cycle:
Neonatal maternal separation augments baseline corticosterone
Plasma corticosterone levels of NMS rats were greater than those measured in controls (Fig. 1; NMS effect: p = 0.006). This increased was observed even though rat pups recovered for nearly 24 h after the last NMS protocol. This result was observed in both males and females (sex effect: p = 0.88).
Body weight and baseline respiratory data
At P12, mean body weights were similar between groups and sex-related differences were not significant (Table 1; NMS effect: p = 0.21; sex effect: p = 0.17). Expiratory duration (Te) of NMS pups was greater than
Discussion
By showing that NMS promotes respiratory instability in 12 days old pups, this study further highlights the significant and profound impact of early life exposure to a non-respiratory stress on the neural circuits that regulate breathing. At this age, the stress-related increase in respiratory variability was greater than in adults (Kinkead et al., 2009) but the sex-specific enhancement of the HVR that characterises adult male rats was not apparent (Genest et al., 2004). Since increased
Acknowledgements
The authors would like to thank Dr. Aida Bairam for insightful discussions and commenting earlier versions of this manuscript. We are also grateful to Evelyne Vachon, animal care specialist, for her assistance with the rat colony. This research was supported by the Canadian Institutes of Health Research and the Canada Research Chair in Respiratory Neurobiology.
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