Neonatal stress increases respiratory instability in rat pups

https://doi.org/10.1016/j.resp.2011.01.014Get rights and content

Abstract

Neonatal maternal separation (NMS) is a form of stress that has persistent, sex-specific effects on respiratory control development. In adult male (but not female) rats, NMS increases the hypoxic ventilatory response and augments respiratory instability during sleep; however, the effects of NMS on respiratory control prior to puberty are unknown. This study tested the hypothesis that NMS augments respiratory instability and the O2 chemosensitivity in 12 days old rats. Pups subjected to NMS were placed in an incubator 3 h/day from P3 to P12. Controls were undisturbed. Breathing and apnea index were measured in normoxia with plethysmography. The ventilatory chemoreflex was assessed by measuring the increase in breathing frequency upon brief exposure to a hypoxic challenge (FIO2=0.09; 5 min). In normoxia, NMS augments the coefficient of variation (CV; an index of respiratory instability) of respiratory frequency and the number of apneas; this effect was more pronounced in male pups. We conclude that disruption of respiratory regulation by NMS is already apparent at P12. Based on results showing that plasma corticosterone levels of NMS pups were still elevated one day after the last NMS episode and the lack of effect of NMS on the O2 chemoreflex index, we propose that disruption of hypothalamic regulation contributes to respiratory instability in NMS 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.

References (78)

  • P. Kc et al.

    Modulation of cardiorespiratory function mediated by the paraventricular nucleus

    Respir. Physiol. Neurobiol.

    (2010)
  • P. Kc et al.

    Paraventricular vasopressin-containing neurons project to brain stem and spinal cord respiratory-related sites

    Respir. Physiol. Neurobiol.

    (2002)
  • M.C.K. Khoo

    Determinants of ventilatory instability and variability

    Respir. Physiol.

    (2000)
  • R. Kinkead et al.

    Neonatal maternal separation and neuroendocrine programming of the respiratory control system in rats

    Biol. Psychol.

    (2010)
  • C.M. Kuhn et al.

    Responses to maternal separation: mechanisms and mediators

    Int. J. Dev. Neurosci.

    (1998)
  • R.M. Lechan et al.

    The TRH neuron: a hypothalamic integrator of energy metabolism

  • S. Levine

    Developmental determinants of sensitivity and resistance to stress

    Psychoneuroendocrinology

    (2005)
  • M.C. Logsdon et al.

    The impact of postpartum depression on mothering

    J. Obstet. Gynecol. Neonat. Nur.

    (2006)
  • S. Ma et al.

    Chronic intermittent hypoxia sensitizes acute hypothalamic–pituitary–adrenal stress reactivity and Fos induction in the rat locus coeruleus in response to subsequent immobilization stress

    Neuroscience

    (2008)
  • S. Macri et al.

    Developmental plasticity of HPA and fear responses in rats: a critical review of the maternal mediation hypothesis

    Horm. Behav.

    (2006)
  • S.G. Matthews

    Antenatal glucocorticoids and the developing brain: mechanisms of action

    Semin. Neonatol.

    (2001)
  • W.B. Mendelson et al.

    Periodic cessation of respiratory effort during sleep in adult rats

    Physiol. Behav.

    (1988)
  • M.L. Nock et al.

    Relationship of the ventilatory response to hypoxia with neonatal apnea in preterm infants

    J. Pediatr.

    (2004)
  • F.L. Powell et al.

    Time domains of the hypoxic ventilatory response

    Respir. Physiol.

    (1998)
  • C.R. Pryce et al.

    Long-term effects of early-life environmental manipulations in rodents and primates: potential animal models in depression research

    Neurosci. Biobehav. Rev.

    (2005)
  • G.M. Renard et al.

    Sexual dimorphism in rats: effects of early maternal separation and variable chronic stress on pituitary-adrenal axis and behavior

    Int. J. Dev. Neurosci.

    (2007)
  • P.J. Schaeffer

    The development of the ventilatory response to cold in very young rats

    Comp. Biochem. Physiol.—Part A: Mol. Integr. Physiol.

    (1998)
  • J.M. Simpson

    Infant stress and sleep deprivation as an aetiological basis for the sudden infant death syndrome

    Early Hum. Dev.

    (2001)
  • H.A. Slotten et al.

    Long-lasting changes in behavioural and neuroendocrine indices in the rat following neonatal maternal separation: gender-dependent effects

    Brain Res.

    (2006)
  • J.M. Szewczak et al.

    Open-flow plethysmography with pressure-decay compensation

    Respir. Physiol. Neurobiol.

    (2003)
  • D.L. Van den Hove et al.

    Prenatal stress and neonatal rat brain development

    Neuroscience

    (2006)
  • E.R. Yeh et al.

    The paraventricular nucleus of the hypothalamus influences respiratory timing and activity in the rat

    Neurosci. Lett.

    (1997)
  • J.M. Abu-Shaweesh et al.

    Neonatal apnea: what's new?

    Pediatr. Pulmonol.

    (2008)
  • R.W. Bavis et al.

    Long-term effects of the perinatal environment on respiratory control

    J. Appl. Physiol.

    (2008)
  • J.M. Bissonnette

    Mechanisms regulating hypoxic respiratory depression during fetal and postnatal life

    Am. J. Physiol. Regul. Integr. Comp. Physiol.

    (2000)
  • V. Cardot et al.

    Ventilatory response to a hyperoxic test is related to the frequency of short apneic episodes in late preterm neonates

    Pediatr. Res.

    (2007)
  • J.L. Carroll

    Plasticity in respiratory motor control: invited review: developmental plasticity in respiratory control

    J. Appl. Physiol.

    (2003)
  • S.E. Davis et al.

    Postnatal developmental changes in CO2 sensitivity in rats

    J. Appl. Physiol.

    (2006)
  • J.A. Dempsey et al.

    Pathophysiology of sleep apnea

    Physiol. Rev.

    (2010)
  • Cited by (26)

    View all citing articles on Scopus
    View full text