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Pain assessment is challenging in neonates. Behavioral and physiological pain scales do not assess neocortical nociception, essential to pain encoding and central pain pathway development. Functional near-infrared spectroscopy (fNIRS) can assess neocortical activation to noxious stimuli from changes in oxy-(HbO) and total-hemoglobin concentrations (HbT). This study aims to assess fNIRS nociceptive functional activation in the prefrontal cortex of neonates undergoing circumcision through changes in HbO and HbT, and the correlation between changes in fNIRS and Neonatal Infant Pain Scale (NIPS), a behavioral pain assessment scale.In healthy term neonates, HbO, HbT, and NIPS were recorded during sequential circumcision events 1-Prep before local anesthetic injection; 2-Local anesthetic injection; 3-Prep before incision; 4-Oral sucrose; 5-Incision; 6-Gomco (hemostatic device) attached; 7-Gomco twisted on; and 8-Gomco removed. fNIRS and NIPS changes after each event were assessed with Wilcoxon signed-rank test and summarized as median and interquartile range (IQR). Changes in fNIRS vs. NIPS were correlated with Spearman coefficient.In 31 neonates fNIRS increased (median [IQR] µmol/L) with noxious events: Local injection (HbO: 1.1 [0.5, 3.1], p .001; HbT: 2.3 [0.2, 7.6], p .001), Gomco attached (HbO: 0.7 [0.1, 1.7], p = .002; HbT: 0.7 [-0.2, 2.9], p = .02), and Gomco twisted on (HbO: 0.5 [-0.2, 1.7], p = .03; HbT: 0.8 [-0.1, 3.3], p = .02). fNIRS decreased with non-noxious event: Prep before incision (HbO: -0.6 [-1.2, -0.2] p .001; HbT: -1 [-1.8, -0.4], p .001). Local anesthetic attenuated fNIRS increases to subsequent sharp stimuli. NIPS increased with subsequent sharp stimuli despite local anesthetic. Although fNIRS and NIPS changed in the same direction, there was not a strong correlation between them.During neonatal circumcision, changes in fNIRS differed between different types of painful stimuli, which was not the case for NIPS, suggesting that fNIRS may complement NIPS to assess the quality of pain.

In the adult, immune and neural processes jointly modulate pain. During development, both are in transition and little is known about the role that the immune system plays in pain processing in infants and children. The objective of this study was to determine if inhibition or augmentation of the immune system would alter pain processing in the infant rat, as it does in the adult. In Experiment 1, rat pups aged 3, 10, or 21 (PN3, PN10, and PN21) days of age were pretreated with NS398 (selective cyclooxygenase (COX)-2 inhibitor) or SC560 (selective COX-1 inhibitor) and tested in the intraplantar formalin test to assess effects of COX inhibition on nociception. Neither drug had an effect on the behavioral response at PN3 or PN10 pups but both drugs attenuated nociceptive scores in PN21 pups. cFos expression in the spinal cord likewise was reduced only at PN21. In Experiment 2, pups were injected with lipopolysaccharide (LPS) prior to the formalin test at PN3 or PN21. LPS increased the nociceptive response more robustly at PN21 than at PN3, while increasing cytokine mRNA equally at both ages. The augmentation of pain responding at PN21 was largely during the late stages of the formalin test, as reported in the adult. These data support previous findings demonstrating late maturing immune modulation of nociceptive behaviors.

Pain is a serious problem for infants and children and treatment options are limited. Moreover, infants born prematurely or hospitalized for illness likely have concurrent infection that activates the immune system. It is now recognized that the immune system in general and glia in particular influence neurotransmission and that the neural bases of pain are intimately connected to immune function. We know that injuries that induce pain activate immune function and suppressing the immune system alleviates pain. Despite this advance in our understanding, virtually nothing is known of the role that the immune system plays in pain processing in infants and children, even though pain is a serious clinical issue in pediatric medicine. This brief review summarizes the existing data on immune-neural interactions in infants, providing evidence for the immaturity of these interactions.

Background Stimulation of a variety of brain sites electrically or by opiates activates descending inhibitory pathways to attenuate noxious input to the spinal cord dorsal horn and produce analgesia. Analgesia induced by electrical stimulation of the periaqueductal gray of the midbrain (PAG) or medial rostral ventral medulla (RVM) matures late, towards the end or past the preweaning period. Descending facilitation takes precedence over inhibition. Yet opiates injected ICV or directly into the PAG induce analgesia relatively early in development. Our goal was to reexamine the role of opiates specific to individual receptor types in analgesia at several supraspinal sites.

Adult central nervous system (CNS) neurons do not regenerate severed axons unaided but may regenerate axons into apposed predegenerated peripheral nerve grafts (PNGs). We examined gene expression by using microarray technology in laser-dissected lateral vestibular (LV) neurons whose axons were severed by a lateral hemisection at C3 (HX) and in lateral vestibular nucleus (LVN) neurons that were hemisected at C3 and that received immunosuppression with cyclosporine A (CsA) and a predegenerated PNG (termed I-PNG) into the lesion site. The results provide an expression analysis of temporal changes that occur in LVN neurons in nonregenerative and potentially regenerative states and over a period of 42 days. Axotomy alone resulted in a prolonged change in regulation of probe sets, with more being upregulated than downregulated. Apposition of a PNG with immunosuppression muted gene expression overall. Axotomized neurons (HX) upregulated genes commonly associated with axonal growth, whereas axotomized neurons whose axons were apposed to the PNG showed diminished expression of many of these genes but greater expression of genes related to energy production. The results suggest that axotomized LVN neurons express many genes thought to be associated with regeneration to a greater extent than LVN neurons that are apposed to a PNG. Thus the LVN neurons remain in a regenerative state following axotomy but the conditions provided by the I-PNG appear to be neuroprotective, preserving or enhancing mitochondrial activity, which may provide required energy for regeneration. We speculate that the graft also enables sufficient axonal synthesis of cytoskeletal components to allow axonal growth without marked increase in expression of genes normally associated with regeneration. J. Comp. Neurol. 519:3433–3455, 2011. © 2011 Wiley-Liss, Inc.

Estrogen modulates pain perception but how it does so is not fully understood. The aim of this study was to determine if estradiol reduces nociceptive responses in part via hypothalamic-pituitary-adrenal (HPA) axis regulation of cyclooxygenase (COX)-1/COX-2 activity. The first study examined the effects of estradiol (20%) or vehicle with concurrent injection nonsteroidal antiinflammatory drugs (NSAIDs) on formalin-induced nociceptive responding (flinching) in ovariectomized (OVX) rats. The drugs were ibuprofen (COX-1 and COX-2 inhibitor), SC560 (COX-1 inhibitor), or NS398 (COX-2 inhibitor). In a second study, estradiol's effects on formalin-induced nociception were tested in adrenalectomized (ADX), OVX, and ADX+OVX rats. Serum levels of prostaglandins (PG) PGE(2) and corticosterone were measured. Estradiol significantly decreased nociceptive responses in OVX rats with effects during both the first and the second phase of the formalin test. The nonsteroidal antiinflammatory drugs (NSAIDs) did not alter nociception at the doses used here. Adrenalectomy neither altered flinching responses in female rats nor reversed estradiol-induced antinociceptive responses. Estradiol alone had no effect on corticosterone (CORT) or prostaglandin levels after the formalin test, dissociating the effects of estradiol on behavior and these serum markers. Ibuprofen and NS398 significantly reduced PGE2 levels. CORT was not decreased by OVX surgery or by estradiol below that of ADX. Only IBU significantly increased corticosterone levels. Taken together, our results suggest that estradiol-induced antinociception in female rats is independent of COX activity and HPA axis activation.

Human infants are often exposed to opiates chronically but the mechanisms by which opiates induce dependence in the infant are not well studied. In the adult the brain regions involved in the physical signs of opiate withdrawal include the periaqueductal gray area, the locus coeruleus, amygdala, ventral tegmental area, nucleus accumbens, hypothalamus, and spinal cord. Microinjection studies show that many of these brain regions are involved in opiate withdrawal in the infant rat. Our goal here was to determine if these regions become metabolically active during physical withdrawal from morphine in the infant rat as they do in the adult. Following chronic morphine or saline treatment, withdrawal was precipitated in 7-day-old pups with the opiate antagonist naltrexone. Cells positive for Fos-like immunoreactivity were quantified within select brain regions. Increased Fos-like labeled cells were found in the periaqueductal gray, nucleus accumbens, locus coeruleus, and spinal cord. These are consistent with other studies showing that the neural circuits underlying the physical signs of opiate withdrawal are similar in the infant and adult.

Microarrays are one of several technologies that allow for measurement the expression of thousands of genes simultaneously. This technological advance provides a challenge for the analysis of these data. In this review we discuss these analytical issues from the initial quality control to normalization, differential expression, clustering and finally functional pathway analysis. We focus on Affymetrix data but many of the issues are the same for other array platforms.

Glutamate receptors, especially N-methyl-D-aspartate (NMDA) receptors, are hypothesized to play key roles in opiate tolerance and withdrawal. There is also accumulating evidence that a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists and group II metabotropic glutamate receptor (mGluR) agonists attenuate opiate withdrawal. However, most existing data are derived from adult animal models. Glutamate receptor types undergo dramatic developmental changes during early life. Thus, the pharmacological effects on opiate withdrawal of NMDA receptor, AMPA receptor, and mGluR antagonists in the developing organism may not be comparable to those in the adult. Indeed, NMDA receptor antagonists do not block morphine tolerance or withdrawal in the 7-day-old rat, but are partially effective in the 14-day-old, and fully effective in the 21-day-old. Thus, there is a transition period around the second post-natal week for NMDA receptor antagonists to suppress opiate tolerance and withdrawal. A combination of in vivo and in vitro assays was used in the present studies to test the effect of drugs acting on AMPA and group II mGlu receptors on morphine withdrawal in rats at 7, 14, and 21 days of age. These ages represent the critical periods when various glutamate receptor subunits undergo differential change. In contrast to NMDA receptor antagonists’ early ineffectivness in suppressing morphine withdrawal, the AMPA receptor antagonist and the group II mGluR agonists were effective at all ages tested. Thus, for the human infant patient, pharmacotherapies to reduce opiate tolerance and withdrawal should focus on non-NMDA ionotropic and metabotropic receptors. # 2004 ISDN. Published by Elsevier Ltd. All rights reserved.

We examined how the experience of a threatening stimulus alters subsequent behavior in a situation where the immediate threat is absent. A small huddle of 12-day-old rats was exposed to a potentially infanticidal adult male rat for 5 min. During male exposure, pups were significantly more immobile than control pups. Thirty, 60, and 180 min after male exposure, the pups were isolated for 5 min from litter and dam in an unfamiliar environment. When isolated, pups that had been previously exposed to the male emitted significantly fewer ultrasonic vocalizations than controls, but did not differ in immobility. Low levels of vocalization were apparent 30 and 60 min after male exposure and were not evident at 180 min. The pups seemed to have adjusted their behavior to a potential male threat in a different context for a limited period of time.

Morphine injected locally to the paw of an adult or an infant rat is analgesic. Opiates specific to micro and kappa opioid receptors, and less consistently to delta opioid receptors, given locally to the site of injury in adult animals are also analgesic in a variety of models of inflammatory pain. To determine which opioid receptor(s) are involved in local analgesia in the immature animal, agonists specific for micro, kappa, and delta opioid receptors were injected into the intraplantar pad in infant rats and the resultant nociceptive behavior and Fos expression assayed in the formalin test. The kappa opioid receptor agonist U50,488 reduced nociceptive behavior in both phases of the formalin test and reduced Fos expression in the dorsal horn of the lumbar spinal cord, at 3 and 21 days of age. Morphiceptin (micro opioid agonist) was analgesic in the 21-day-old pups, but not the 3-day-old pups, measured behaviorally or by Fos expression. DPDPE (delta opioid agonist) was not analgesic at either age. We also tested the effects of opioid receptor antagonists on morphine's local analgesic action. Naltrexone, and to a lesser extent the micro opioid antagonist CTOP, antagonized morphine's analgesic effect. Kappa and delta opioid receptor blockers were inactive. The results demonstrate the ability of the kappa opioid system to mediate analgesia in the neonate at the site of injury in acute and chronic pain models, that the micro opioid agonists are active later in development, but that morphine is analgesic in part through micro opioid receptors.

During early ontogeny, stimuli that pose a threat to an animal change. Unrelated adult male rats may kill young rats, but infanticide ends around weaning. Predation, on the other hand, may increase during early ontogeny when rats begin to extend their activity range. We investigated the developmental course of two defensive responses, immobility and analgesia, in young rats exposed to an adult male rat or to predator cues. Preweaning 14-day-old rats became immobile and analgesic when exposed to the male and showed immobility but not analgesia when exposed to cat odor. On Day 26, around weaning, the presence of the male rat no longer induced immobility and analgesia whereas cat odor produced higher levels of immobility and analgesia compared to control and male-exposed animals. This developmental change in responsivity may reflect the differences in the risk of being harmed by a male or a cat during different periods of ontogeny.

Opiate withdrawal behaviors in the infant differ from those of the adult. The neural circuitry underlying opioid withdrawal in the adult rat is well defined and includes the locus coeruleus (LC) and periaqueductal gray (PAG), with a minor role of the amygdala. Because the different behaviors that constitute the infant syndrome may be mediated by different neural circuits, we tested the hypothesis that these three sites are involved in opiate withdrawal. Pups were injected with morphine from day 1- 6 after birth (b.i.d.) and on the morning of the seventh day. Withdrawal was then elicited by local injection of the opioid antagonist methylnaloxonium into the LC, PAG, or amyg- dala. Withdrawal signs were precipitated in a dose-dependent manner following injec- tion into the LC or PAG, but not the amygdala. The withdrawal behaviors elicited from the LC and PAG included both the same and different behaviors. The results support the hypothesis that the neural circuitry mediating opiate withdrawal behaviors is similar in infant and adult animals, but the behaviors expressed are age-specific. Synapse 39: 139 -151, 2001. © 2001 Wiley-Liss, Inc.

Administration of NK1 antagonists in adult animals attenuates the nociceptive response in the formalin test, indicating that the neurokinins and the NK1 receptor play a role in mediating this pain response. The number and distribution of NK1 receptors change dramatically during development, and the age at which they become involved in pain processing is not known. We examined the role of NK1 receptors in the formalin model in rats ranging in age between 3- and 21-days old. An NK1 antagonist, CP99,994, and its less active enantiomer CP100,263 were administered to the spinal cord (intrathecal), systemically (subcutaneous), or locally (intraplantar). Intrathecal administration of CP99,994, but not CP100,263, attenuated pain behaviors in the second phase of the formalin response in 14-day and 21-day old rats, but did not alter the pain response in 3-day or 10-day old rats. CP99,994 also reduced the expression of the c-fos protein in the superficial dorsal horn of 21-day old rats. Systemic and intraplantar injection of either CP99,994 or CP100,263 reduced the pain response to formalin in 3-day and 21-day old rats, suggesting a non-NK1 mediated mechanism of action. These results indicate that, within the spinal cord, NK1 receptors start to play a role in the pain response to formalin between 10 and 21 days. Moreover, analgesia induced by systemic or local injection of NK1 antagonists involves mechanisms other than, or in addition to, the NK1 receptor.

Endomorphin-1 and endomorphin-2 are recently described peptides with high affinity and specificity for the mu opioid receptor. They are believed to be the endogenous ligands for that receptor. We describe the maturation of the endomorphins in brain and spinal cord using recently characterized antibodies to each. Endomorphin-1-like immunoreactivity was examined in brain, focusing on the periaqueductal gray of the midbrain and the diagonal band of Broca; endomorphin-2-like immunoreactivity is reported for the medulla and spinal cord. In these regions, and in all other regions studied but not described in this paper, the endomorphins were not seen at birth or at 3 days of age. Staining was present in 7-day-old and older animals. At these early ages, the pattern and density of staining are not fully developed, but appear complete by 21 days of age. The results suggest that both endomorphin-1 and endomorphin-2 develop relatively late compared to other opioid peptides.

Previous research examining the ability of neonatal rats to adapt to repeated isolation demonstrated that an opioid-dependent decline in ultrasonic vocalizations occurred across a series of isolations (Goodwin, Molina, & Spear, 1994). These findings were expanded in the present study. In the first experiment, the decline in vocalization rates was found to result from the release of endogenous opioids throughout the series of isolations. Although naltrexone attenuated the decline in calling rates relative to vehicle-treated subjects, there was still a significant decline in calling rates following opioid receptor blockade. In the second experiment, two injections of naltrexone did not attenuate the decline in calling rates any more than a single injection did, suggesting that there must also be some nonopioid process that modulates this decline. In both experiments, activity levels and, in the first experiment, the amount of body heat lost in the repeatedly isolated subjects declined in a nonnaltrexone reversible manner. In a final study, after calling rates had been suppressed by a series of isolations, a brief exposure to the mother was found to restore baseline calling rates, suggesting the decline is not the consequence of fatigue. The attenuation of vocalization rates, activity, and loss of body heat are adaptive responses of infant rats to isolation; however, of these three, only the attenuation of vocalization rates is consistently modulated by the release of endogenous opioids.

Despite the importance of pediatric anesthesiology, the sites and mechanisms of anesthetic action in the neonate are not well described in either human or nonhuman species. This experiment investigated suppression produced by different anesthetic agents of neuronal activity in the lumbar spinal cord of the 3-day-old rat. The expression of the c-fos immediate early gene following formalin injection into the hindpaw was used as a marker for neuronal activity. Pups were anesthetized by one of the following often-used agents: methoxyflurane, acepromazine, a mixture of ketamine and xylazine, and hypothermia. All treatments induced behavioral anesthesia. Despite the behavioral anesthesia, the ketamine-xylazine mixture was completely ineffective in suppressing formalin-induced-Fos expression. In contrast, methoxyflurane and hypothermia blocked the appearance of the Fos protein. Similarly, acepromazine was effective in eliminating some of the Fos-labeled nuclei. These data suggest that, in the infant rat, both hypothermia and methoxyflurane act in part at the spinal level by depressing either primary afferents or dorsal horn neuronal activity whereas the site of action for ketamine-xylazine may be located supraspinally.

This study examined the normal development of neuronal activity in the suprachiasmatic nuclei (SCN) of rats between age 3-60 days, using Fos protein-like immunoreactivity (Fos-LI) as a marker. At age 3 days, Fos-positive nuclei are sparsely distributed throughout the SCN. Between age 3-10 days, the density of labeled nuclei increases significantly. Fos-LI labeling is maximal at 10 days. Between age 10-14 days, the number of labeled nuclei decreases and remains relatively constant thereafter, although the intensity of the reaction product diminishes as the animal matures. By age 60 days, the number of Fos-LI labeled nuclei in the SCN is substantially decreased and is essentially the same as in the 3-day-old rat. The appearance of Fos-LI nuclei in the SCN during development appears to reflect the development of visual system afferents to the nucleus as well as the development of intrinsic SCN synaptology.