Repeated neonatal pain influences maternal behavior, but not stress responsiveness in rat offspring

Abstract

Early preterm neonates in the Neonatal Intensive Care Unit (NICU) are subjected to repeated painful procedures which could sensitize their responses to pain and potentiate neuroendocrine and behavioral responses to subsequent stressors in the long-term. In this study, we used the model of the neonatal rat to test the effects of repeated pain during the first 2 weeks of life on neuroendocrine responses (CRF, ACTH and corticosterone) to stressors varying in intensity and on maternal behavior in the postnatal period. To closely mimic the type of repeated painful stimulus experienced by preterm neonates (i.e., heelstick), neonatal rats aged day 2–14 were submitted daily to having their rear heels warmed to 34 °C and pricked (handled and pain, HP) or not (handled, H) with a needle. For the procedure, all pups were separated from their mothers for a total period of 15 min and reunited afterwards. Unhandled (UH) pups not subjected to daily maternal separation were used as controls. On days 6 and 12, litters from the HP and H groups were videotaped for 90 min upon return with the mother and maternal behavior was analyzed. Frequency of ultrasonic vocalizations (USV) were recorded during the procedure and upon return of pups with the mother. On day 15 and 20, rat pups from all groups were exposed to a 3-min ether vapor stressor or to an openfield for 10 min. Plasma ACTH and corticosterone concentrations were determined at 0, 5, 30, 60 and 120 min after stress onset. Our results show that repeated pain did not modify body weight of the pups, however, on day 6 of life, maternal pup grooming was increased significantly (P<0.05) in the HP group compared to the H group. Frequency of USV was not changed between H and HP rats either during the separation or after reunion with the mother. Plasma ACTH and corticosterone levels under basal or stimulated conditions were not different between UH, H and HP groups. However, the UH pups showed a tendency towards higher ACTH secretion after stress compared to H and HP groups. These results suggest that repeated pain during the first 2 weeks of life in the rat does not lead to significant changes in stress responsiveness in 2-week-old pups, but we suggest that changes in mother–pup interaction (increased grooming) might act as a buffer on the cumulative effect of pain on stress responsiveness.

Introduction

Early preterm neonates in the NICU (Neonatal Intensive Care Unit) are routinely subjected to a number of invasive procedures having a strong pain component without benefit of analgesia [1], [5], [22], [25]. Although there have been reports of diminished responsiveness over days or weeks in relationship to higher frequency of painful procedures [16], [17], [22], [26], the effect of these repeated painful procedures on subsequent stress responsiveness and homeostasis are not known. Pain in infants is considered as a potent stressor eliciting physiological and behavioral responses that compare well with responses observed in adults [47]. Early exposure to stressors during perinatal and neonatal development is known to have long-term consequences on the ability of the organism to cope with stress [9], [31], [39], [40] and the susceptibility to develop disease in adulthood [34], [43]. The maximal window of susceptibility for the environmental ‘programming’ of brain function and stress responsiveness in rodents has been identified to span the perinatal period and in particular, the first 2 weeks of life [38]. During this period, procedures such as maternal deprivation or neglect, handling, or repeated maternal separation have all been demonstrated to alter the activity of the HPA axis and several behavioral functions in adulthood (for a review see Ref. [51]). Recent studies have also demonstrated the critical impact of maternal behavior and specific features of maternal care on the development of individual differences in stress responsiveness in adulthood [35]. Mothers providing more licking and grooming to their pups during the first weeks of life were shown to enhance hippocampal development and function in their adult offsprings [32]. Alternatively, lack of handling during infancy has been known to induce higher cortisol secretion and cognitive deficits in children [10]. Taken together, these studies suggest that environmental factors modifying the neonatal physiology (e.g., stress) and/or maternal behavior might be equally important to determine adult predisposition to stress-related diseases. This period in the neonatal rat is also critical for the maturation of pain neurotransmission since segmental control mechanisms within the spinal cord and descending control of pain transmission from higher CNS regions are not mature until after the second week of life [13], [14]. In terms of development of pain pathways, it is believed that the first 2 postnatal weeks of development in the rat correspond well to a gestational period of 26–32 weeks in the human neonate [13]. Other aspects of brain development such as hippocampal formation and synaptogenesis display a different ‘equivalence’ between humans and rodents as outlined in a recent review by Avishai-Eliner et al. [4]. The neonatal rat provides a unique opportunity to determine the impact of repeated pain on subsequent activity of the hypothalamus–pituitary–adrenal (HPA) axis as well as sensitization of pain and stress responses. Various stressors with a pain component stimulate ACTH secretion during the neonatal period in the rat [45], [49], [50] and we have previously demonstrated that repeated exposure to stress potentiate ACTH and corticosterone responses in 10-day-old pups [48]. Similarly, sensitization of pain responses have been described until 32 weeks gestation in infants and 2 weeks in rat pups, followed by habituation thereafter [24]. These results suggest that repeated pain stress could lead to a potentiated neuroendocrine response in both neonatal babies and rat pups.

The present study was designed to determine whether potentiation of neuroendocrine responses to stress occur during neonatal life in rat pups submitted to repeated pain in infancy and to determine whether changes in maternal behaviour towards litters repeatedly exposed to pain could have beneficial effects on stress responsiveness. Our results show that repeated pain during the first 2 weeks of life in rats modified some aspects of maternal behaviour without affecting hormonal stress responses in preweaning rats. Increased maternal pup grooming and the presence of littermates at the time of the painful procedure is thought to act as a comfort measure modifying endocrine responses.