Long-term effects of neonatal surgery on adulthood pain behavior

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Abstract
The long-term consequences of neonatal noxious stimulation on adulthood pain behavior were investigated in male and female mice. On the day of birth, mouse pups were exposed to a laparotomy under cold anesthesia followed by an analgesic dose of morphine (10 mg/kg) post-operatively, or a saline control. An additional group of subjects was exposed to the non-noxious aspects of the surgical procedure (cold exposure, separation from the dam, injection) comprising a ‘sham’ surgery control group, whereas another group of control subjects was administered an injection of saline or morphine, but was otherwise undisturbed. Behavioral observations of the pups immediately following the procedure indicated that the laparotomy produced increased distress vocalizations in the ultrasonic range (40 kHz) compared to both groups of control subjects. During 90 min observations periods following the surgery and 1-week later, maternal care did not vary among treatment conditions. In adulthood, offspring were tested for nociceptive sensitivity on the hot-plate (HP; 53 °C), tail-withdrawal (TW; 50 °C) and acetic acid abdominal constriction test (AC). On both the TW and the AC tests, neonatal surgery decreased pain behavior relative to both groups of control subjects, an effect that was reversed by post-operative morphine treatment. On the HP test, both groups of subjects exposed to the stressful aspects of neonatal surgery (laparotomy or sham surgery) exhibited decreased pain behavior in adulthood. These findings suggest that early exposure to noxious and/or stressful stimuli may induce long-lasting changes in pain behavior, perhaps mediated by alterations in the stress-axis and antinociceptive circuitry. Keywords Neonatal surgery Tail-withdrawal Hot-plate Abdominal constriction 1 Introduction Pain pathways are formed and functional at birth in humans and in laboratory animals ( Anand, 2000, 2001; Fitzgerald and Beggs, 2001; Fitzgerald and Gibson, 1984; Garg et al., 2003; Johnston et al., 1993; McLaughlin et al., 1990 ). Clinicians observe behavioral and physiological responses to routine noxious stimuli (e.g. needle sticks) in human infants ( Grunau et al., 1990; McCulloch et al., 1995 ), and neonatal intensive care often involves painful invasive procedures. Thus, neonatal pain experience may contribute to variability in adult sensitivity to noxious stimuli. In rats, early injury leads to hyperinnervation of the injured area that persists into adulthood. Unilateral application of Complete Freund's Adjuvant (CFA) to the hindpaw causes long-lasting increases in adult pain behavior following re-inflammation, with increased density and lowered activation thresholds of ipsilateral superficial dorsal horn terminals ( Peng et al., 2003; Ruda et al., 2000; Tachibana et al., 2001 ). In this model, basal thermal nociception is unaffected by the neonatal noxious stimulus ( Ruda et al., 2000 ), although ipsilateral dorsal horn neurons display heightened responsiveness to noxious thermal stimuli applied to the affected paw in adulthood ( Peng et al., 2003 ). In contrast, formalin injection applied to all four paws daily for the first post-natal week leads to long-lasting thermal hypo sensitivity of the hindpaw ( Anand et al., 1999; Bhutta et al., 2001 ). Carrageenan inflammation of the hindpaw similarly leads to a long-lasting lowering of sensitivity to thermal noxious stimuli in the affected paw, but also causes hypersensitivity to thermal stimuli in the presence of ongoing inflammation following adulthood exposure ( Lidow et al., 2001 ). These disparate findings have led some to conclude that neonatal inflammation (of moderate intensity) leads to both basal hyposensitivity in the affected region, and hypersensitivity to re-inflammation or injury in adulthood ( Lidow, 2002; Ren et al., 2004 ). These findings extend to a non-inflammatory noxious stimulus (neonatal footshock), which similarly leads to a diminished response to noxious thermal stimuli applied in adulthood ( Bernardi et al., 1986; Shimada et al., 1990 ). Few investigations, however, have assessed overall nociceptive sensitivity (i.e. away from the site of injury) in adults exposed to locally applied neonatal noxious stimuli. It has been observed that some of the procedures that are used to model neonatal pain experience are unusually severe in their intensity and chronicity ( Lidow, 2002 ). Although some clinically relevant neonatal procedures have been employed in animal studies (e.g. neonatal skin wounding, short-duration inflammation, repeated needle sticks), a more realistic animal model of neonatal pain experience could prove informative for understanding long-lasting alterations. The purpose of the current study was three-fold. First, we wished to assess the long-term alterations in pain behavior following neonatal abdominal surgery, a clinically relevant noxious stimulus. Second, we assessed adulthood alterations in nociceptive sensitivity at body loci other than the site of injury. Lastly, we attempted to extend the literature on long-term effects of early noxious stimulation from the rat (the species used in all previous studies) to the laboratory mouse, since we expect future investigations to be concerned with the interaction between early noxious stimulation and genetic profile. 2 Methods 2.1 Subjects The subjects in this study were the offspring of breeding pairs of outbred CD-1 ® mice, bred in our laboratory (breeders obtained from Harlan Sprague–Dawley, Indianapolis, IN). Animals were housed in a light- (12:12 h, lights on at 08:00) and temperature (20 °C)-controlled environment with free access to food (Harlan Teklad 8604) and tap water. Following weaning at 3 weeks of age, subjects were housed with same-sex littermates in groups of 2–6 in polypropylene cages until adult testing (7–12 weeks of age). All procedures were reviewed and approved by the Haverford College Animal Care and Use...