Repetitive acute pain in infancy increases anxiety but does not alter spatial learning ability in juvenile mice

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Abstract

We assessed the long-term behavioural effects of a single acute or repetitive inflammatory pain experienced during infancy. Groups of male and female CD1 mice were subjected to either an acute single pain, i.e. a tail clip or sham pain at P8, or acute repetitive pain in the form of needle pricks or sham pain from P8 to P14. All of the subjects were tested in the elevated plus maze at P30 and in the Morris Water Maze from P31 to P38. Mice in the acute single pain and sham groups did not differ on measures of anxiety in the plus maze. Mice in the repetitive pain group demonstrated significantly more anxious behaviours than controls in the elevated plus maze as they spent less time in the open arms, made fewer open arm entries, displayed fewer head dips and showed more stretch attend postures. There were no effects of single or repetitive pain treatments in the latency to find the hidden platform in the Morris Water Maze. Overall, these data suggest that acute repetitive pain experienced during infancy may increase anxiety later in life but it does not influence spatial learning as measured in the Morris Water Maze. The origin of the anxiogenic profile shown in the acute repetitive pain mice may be a result of changes in neural circuitry or context dependent learning and is currently under further investigation with this paradigm.

Introduction

The technical advances that have lead to enhanced survival for acutely ill newborns have been accompanied by an increase in potentially painful diagnostic and treatment procedures. Repeated heel pricks, intravenous drug delivery, endotracheal intubation and neonatal surgeries have become relatively common and often result in acute inflammatory pain in the neonate [5], [18]. The mechanisms which contribute to inflammatory pain have been thoroughly investigated, primarily in preparations from adult animals, and involve alterations in intracellular signalling cascades as well as transcription-related increases in both receptors and neurotransmitters [13], [17], [19], [29]. These responses may lead to short-term functional and structural changes in adult animals [1], [2], [15], [22]. In newborns, these processes are experienced at a time when the brain is extraordinarily plastic with the nervous, endocrine and immune systems all undergoing functional and structural development [9]. Consequently, it has been hypothesised that exposure to painful stimuli during such a critical period could disrupt these neurodevelopmental processes and lead to permanent changes in the brain as well as in behaviour [4], [25].

Several investigators have found long-term changes in response to inflammatory pain in neonatal rats in peripheral nerve terminals and within the spinal cord itself. Reynolds and Fitzgerald [22] assessed peripheral changes following skin wounds in P0, P7, P14 and adult rats. An examination of the axonal sprouting response revealed a long lasting, i.e. 21–42 days, cutaneous hyperinnervation. The innervation density was of a greater magnitude and duration when the wounds were performed in P0 and P7 rats compared with P14 animals. In the latter animals, changes were of a more transient nature similar to the effect found in adults. These results suggest the existence of a critical window for changes to create long-term effects. In the latter study, sensory thresholds were also measured in rats that received skin wounds on P0. They were found to be significantly lower than thresholds in same-age controls for at least 24 days. The authors suggested this provides evidence of pain or hypersensitivity long after the wound has healed.

Recently, a persistent inflammatory pain in neonatal rats resulting from a single injection of Freund’s adjuvant (FA) in P0 and P3 neonatal rats has been shown to be associated with greater density and distribution in spinal cord laminae I and II dorsal afferent axons in adult animals [24]. In contrast, such permanent changes were not demonstrated in adult animals that had previously been injected at P14. Behavioural responses to the initial injection of CFA were not assessed in adult animals. Nonetheless, when injected with formalin, adults that had been treated as neonates at an unspecified age exhibited pain responses earlier than controls during the late phase of the test [24].

To test the hypothesis that such structural changes could be paralleled by long-term behavioural effects, Anand et al. [3] assessed a number of behavioural responses of rat pups exposed to needle pricks daily from P0 to P7. Compared with controls, these rats showed decreased latency to respond to a thermal stimulus prior to weaning. There were no differences in hotplate latencies between adult experimental and control rats. As adults, rats that had received four paw pricks daily had an increased latency and decreased frequency to enter an open field and an increase in alcohol preference. The authors suggested that these behavioural effects in the adult rats subjected to repetitive neonatal pain were a result of increased anxiety. In a follow up study, Bhutta et al. [7] assessed the effects of formalin-induced inflammatory pain on P0 to P7 rats. When tested as adults, both male and female experimental animals had increased hotplate latencies compared with controls and males showed a decreased preference for alcohol. Males but not females showed a decrease in locomotor activity in a behavioural chamber. The different methods used to induce the early inflammatory pain in these experiments may have resulted in the mixed support for the hypothesis that early repetitive pain leads to increased anxiety in adulthood.

Anand et al. [3] also determined that following repeated paw pricks as neonates adult rats recognised familiar conspecifics in a social discrimination task for a longer period of time than controls. This latter result is in agreement with other work that suggests that memory and/or performance is enhanced by a previous painful experience. Neonatal rats exposed to thermal pain had better performance on a two-way active avoidance task than control animals [6]. Similarly, young rats that had experienced foot shocks mastered a lever pressing task faster than controls [16].

The experiments thus far have reported physical and behavioural effects in rats (P0–P7) that are developmentally equivalent to preterm infants [10]. In this study, we have used a different procedure to determine if an early pain experience in older neonatal mice also results in long-term behavioural changes. We exposed P8 mice to a single event of acute pain in the form of a tail clip. A second group of mice were given daily paw pricks (P8–P14) on the dorsal surface of a front and contralateral rear paw. We choose to use animals of these ages as the maturity of an 8-day-old rodent has been reported to be comparable to that of a full-term infant [10]. We examined the effects of these procedures in P30 to P38 animals to determine if the hypothesised effects would manifest themselves in prepubertal animals. These procedures enabled us to investigate the long-term effects of a fairly typical experience in a full-term human infant.

Given the somewhat conflicting findings as to whether anxiety is affected by early pain [3], [7], we used the elevated plus maze to assess anxiety. This test is known for its high ecological validity and reliable measurement of behaviours associated with anxiety and risk assessment in rodents [23], [27]. To further investigate reports that pain in neonates enhances learning [6], [16], we also assessed spatial learning in the Morris Water Maze.

Section snippets

Animals

CD1 mice (N=56), offspring of adult males and females purchased from Charles River Canada (St. Constant, Quebec) and bred at Dalhousie University, were used in this experiment. The day of birth was designated as Postnatal Day 0 (P0). Litters were culled to a group of four males and four females on P3 and weaned on P21. Prior to weaning, the mice were housed with their dams in polycarbonate cages (30 cm×15 cm×10 cm) with wood-chip bedding. Following weaning, the mice were housed in same sex groups

Elevated plus maze

There were no significant differences between mice that received the single tail cut and sham cut controls in any of the eight behaviours scored in the elevated plus maze (all ts(26)<1.31), Fig. 1 illustrates these results.

Mice that received repetitive paw pricks made fewer open arm entries (t(26)=−3.80, P<0.0008), spent less time in the open arms of the elevated plus maze (t(26)=−3.32, P<0.003), displayed fewer head dips (t(26)=−2.55, P<0.02) and showed more stretch attend postures than the

Discussion

In these experiments, we have demonstrated that infant mice that experience acute repetitive pain from P8 to P14 show a pattern of behaviour in the elevated plus maze as juveniles that is characteristic of anxiety, i.e. a significant increase in stretch attends and a decrease in head dips and time spent in the open arms and frequency of open arm entries. In contrast, mice that experienced a single acute pain on P8 did not show anxious behaviours as juveniles. Mice exposed to either a single or

Acknowledgements

This research was supported by a NSERC operating grant to H.M. Schellinck and a K.M. Hunter Charitable Foundation/CIHR Doctoral research award and the Spatz Doctoral Training Award 2002 (Alzheimer Society of Canada and IA-CIHR) to L.E. Stanford.

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