Recruitment of hypothalamic orexin neurons after formalin injections in adult male rats exposed to a neonatal immune challenge

Exposure to early life physiological stressors, such as infection, is thought to contribute to the onset of psychopathology in adulthood. In animal models, injections of the bacterial immune challenge, lipopolysaccharide (LPS), during the neonatal period has been shown to alter both neuroendocrine function and behavioural pain responses in adulthood. Interestingly, recent evidence suggests a role for the lateral hypothalamic peptide orexin in stress and nociceptive processing. However, whether neonatal LPS exposure affects the reactivity of the orexin system to formalin-induced inflammatory pain in later life remains to be determined. Male Wistar rats (n=13) were exposed to either LPS or saline (0.05mg/kg, i.p) on postnatal days (PND) 3 and 5. On PND 80-97, all rats were exposed to a subcutaneous hindpaw injection of 2.25% formalin. Following behavioural testing, animals were perfused and brains processed for Fos-protein and orexin immunohistochemistry. Rats treated with LPS during the neonatal period exhibited decreased licking behaviours during the interphase of the formalin test, the period typically associated with the active inhibition of pain, and increased grooming responses to formalin in adulthood. Interestingly, these behavioural changes were accompanied by an increase in the percentage of Fos-positive orexin cells in the dorsomedial and perifornical hypothalamus in LPS-exposed animals. Similar increases in Fos-protein were also observed in stress and pain sensitive brain regions that receive orexinergic inputs. These findings highlight a potential role for orexin in the behavioural responses to pain and provide further evidence that early life stress can prime the circuitry responsible for these responses in adulthood

Low formalin concentrations induce fine-tuned responses that are sex and age-dependent: A developmental study

The formalin test is increasingly applied as a model of inflammatory pain using high formalin concentrations (5–15%). However, little is known about the effects of low formalin concentrations on related behavioural responses. To examine this, rat pups were subjected to various concentrations of formalin at four developmental stages: 7, 13, 22, and 82 days of age. At postnatal day (PND) 7, sex differences in flinching but not licking responses were observed with 0.5% formalin evoking higher flinching in males than in females. A dose response was evident in that 0.5% formalin also produced higher licking responses compared to 0.3% or 0.4% formalin. At PND 13, a concentration of 0.8% formalin evoked a biphasic response. At PND 22, a concentration of 1.1% evoked higher flinching and licking responses during the late phase (10–30 min) in both males and females. During the early phase (0–5 min), 1.1% evoked higher licking responses compared to 0.9% or 1% formalin. 1.1% formalin produced a biphasic response that was not evident with 0.9 or 1%. At PND 82, rats displayed a biphasic pattern in response to three formalin concentrations (1.25%, 1.75% and 2.25%) with the presence of an interphase for both 1.75% and 2.25% but not for 1.25%. These data suggest that low formalin concentrations induce fine-tuned responses that are not apparent with the high formalin concentration commonly used in the formalin test. These data also show that the developing nociceptive system is very sensitive to subtle changes in formalin concentrations.Ihssane Zouikr, Melissa A. Tadros, Vicki L. Clifton, Kenneth W. Beagley, Deborah M. Hodgso

Priming of inflammatory pain responses by a neonatal immune challenge: implications of neuroimmune-endocrine communication for pain

Research Doctorate - Doctor of Philosophy (PhD)The perinatal period, which encompasses both in utero and neonatal life, represents a time of significant plasticity during which many physiological systems including the immune, endocrine, and nociceptive systems are undergoing fine-tuning and maturation. Thus, an exposure to environmental stimuli during this sensitive period of development can interfere with the normal developmental trajectory of these physiological systems, leading to maladaptive responses later in life. Several animal and human studies have documented that exposure to a variety of stressors such as psychological, physiological, or social stress, can critically influence how organisms evolve and respond to their environment later in life. One factor that has recently received considerable interest is exposure to bacteria during the neonatal period. Exposure to the bacterial mimetic, Lipopolysaccharide (LPS), is an established model of early life immune-mediated stress. Our laboratory has previously shown that neonatal LPS exposure is associated with altered behavioural, endocrine, and immune responses later in life. However, the impact of neonatal LPS exposure on nociceptive responses later in life is less known. The primary aim of the current thesis was to develop a profile of the formalin-induced behavioural alterations that are associated with neonatal LPS exposure, as well as characterizing the neuroendocrine, neuroimmune, spinal, and supraspinal changes associated with this behavioural profile. To achieve this aim, we have subjected Wistar rats to intraperitoneal administration of LPS (LPS, Salmonella enterica, serotype enteritidis) on postnatal days (PNDs) 3 and 5 (birth = PND 1) and subjected them to formalin injection at PNDs 7, 13, 22, and 80-97. The first manuscript (Zouikr et al., 2013) examined the impact of low formalin concentrations, not previously used in the literature, on formalin-induced nociceptive responses (i.e. flinching and licking) during the first three postnatal weeks in rats. The results indicated that low formalin concentrations (0.3-2.25%) induced developmentally regulated pain responses. The characteristic biphasic nociceptive response appeared as early as PND 13 following an intraplantar injection of 0.8% formalin. Additionally, we demonstrated that PNDs 7, 13, 22 and adult rats displayed fine-tuned responses with low formalin concentrations including appearance of licking responses in one week old rats. Following the optimization of the formalin test, in the second manuscript (Zouikr et al., 2014b), we investigated the behavioural profile of infant and preadolescent rats following exposure to an immune challenge during the neonatal period. We demonstrated for the first time that dual exposure to LPS at PNDs 3 and 5 exerts long-term effects on inflammatory pain responses in a developmentally regulated manner. An increased susceptibility (i.e. hyperalgesia) to formalin-induced licking (at PND 13) and flinching (at PND 22) responses was observed following neonatal LPS exposure. Neonatal LPS exposure did not alter formalin-induced nociceptive response in PND 7 rats. We further characterized the neuroendocrine changes associated with this age-dependent behavioural hyperalgesia. LPS-treated rats displayed an increased plasma corticosterone levels at PND 22, but not PND 13, and a shift in the balance of glucocorticoid and mineralocorticoid receptor mRNA in the hypothalamus at PND 22 following formalin injection. We have also investigated the impact of neonatal LPS challenge on spinal dorsal horn neuronal changes and found significant changes in the intrinsic properties of spinal dorsal horn (SDH) neurons in PND 22 rats after neonatal LPS exposure as indicated by decreased input resistance and decreased Action Potential (AP) amplitude in LPS-treated rats. These data provided the first evidence that neonatal immune challenge produces developmentally regulated changes in formalin-induced nociception, HPA axis function, and SDH neuronal properties. We then focused on the supraspinal changes associated with the behavioural hyperalgesia in preadolescent rats. The third manuscript (Zouikr et al., 2014a) investigated whether the increased formalin-induced behaviour observed in preadolescent rats treated with LPS as neonates is due to decreased neuronal activation of the PAG, a substrate known to mediate analgesia. cFos was used as a marker of neuronal activation. We demonstrated that the LPS-induced hyperalgesia in PND 22 rats was associated with distinct recruitment of supra-spinal regions involved in analgesia as indicated by significantly attenuated Fos-protein induction in the rostral dorsal periaqueductal grey (DPAG) as well as rostral and caudal axes of the ventrolateral PAG (VLPAG). Formalin injections were associated with increased Fos-protein labelling in lateral habenula (LHb) as compared to medial habenula (MHb), however the intensity of this labelling did not differ as a result of neonatal immune challenge. In the fourth manuscript (Zouikr et al., 2014c, in press), we were interested in determining the long-term effects of neonatal LPS challenge on formalin-induced nociceptive behaviour. Specifically, whether the LPS-induced hyperalgesia persists into adulthood. We were also interested in determining and characterizing the neuroimmune alterations to be driven such behavioural, neuroendocrine, spinal, and supraspinal alterations observed in manuscript 2 and 3 (Zouikr et al., 2014a; Zouikr et al., 2014b). The fourth manuscript demonstrated that neonatal LPS exposure induces increased formalin-induced nociceptive behaviour in both preadolescent (i.e. PND 22) and adult rats (i.e. PNDs 80-97). This behavioural hyperalgesia was accompanied by developmentally regulated changes in peripheral and central immune responses as indicated by enhanced plasma levels of IL-1β and enhanced mast cell degranulation in LPS-treated preadolescent rats as well as increased hippocampal IL-1β in LPS-treated adult rats. Taken together, these studies demonstrate that the early microbial environment plays an eminent role in determining inflammatory pain sensitivity later in life via the action on behaviour, immune, neuroendocrine, spinal, and supraspinal systems. Because of the well regulated interaction between all of these physiological systems and their vulnerability during sensitive windows of development, particularly during the neonatal period when the neurocircuitry underlying the nociceptive and the immune system are undergoing significant plasticity, it is important to guarantee a healthy and infection-free environment to vulnerable infants such as preterm infants. This will help reduce possible interference with the normal developmental trajectory of the brain and the nociceptive system. The findings from this thesis also emphasize on the importance of the neuroimmune interface in modulating pain sensitivity. If we are to combat this stubborn condition that is chronic pain, future therapeutic approaches should take into account the critical aspect and the critical role of the neuroimmune interface by targeting component of the immune system (e.g. antagonizing the effects of pro-inflammatory cytokines) in addition to targeting component of the nervous system because these two physiological systems constitute one overarching, highly modulated system

Neuroimmune Interface in Health and Diseases

It is now well appreciated that the immune system, in addition to its traditional role in defending the organism against pathogens, communicate in a well-organized fashion with the brain to maintain homeostasis and regulate a set of neural functions. Perturbation in this brain-immune interactions due to inflammatory responses may lead to psychiatric and neurological disorders. Microglia are one of the essential cells involved in the brain-immune interactions. Microglial cells are now not simply regarded as resident tissue macrophages in the brain. These cells are derived from myeloid progenitor cells in the yolk sac in early gestation, travel to the brain parenchyma and interact actively with neurons during the critical period of neurogenesis. Microglia provide a trophic support to developing neurons and take part in the neural wiring through the activity-dependent synapse elimination via direct neuron-microglia interactions. Altered microglial functions including changes in the gene expression due to early life inflammatory events or psychological and environmental stressors can be causally related to neurodevelopmental diseases and mental health disorders. This type of alterations in the neural functions can occur in the absence of infiltration of inflammatory cells in the brain parenchyma or leptomeninges. In this sense, the pathogenetic state underlying a significant part of psychiatric and neurological diseases may be similar to “para-inflammation”, an intermediate state between homeostatic and classical inflammatory states as defined by Ruslan Medzhitov (Nature 454:428-35, 2008). Therefore, it is important to study how systemic inflammation affects brain health and how local peripheral inflammation induces changes in the brain microenvironment. Chronic pain is also induced by disturbance in otherwise well-organized multisystem interplay comprising of reciprocal neural, endocrine and immune interactions. Especially, early-life insults including exposure to immune challenges can alter the neuroanatomical components of nociception, which induces altered pain response later in life. Recently the discrete roles of microglia and blood monocyte-derived macrophages are being defined. The distinction may be further highlighted by disorders in which the brain parenchymal tissue is damaged. Therefore, studies investigating the dynamics of immune cells in traumatic brain injury and neurotropic viral infections including human immunodeficiency virus, etc. as well as neurodegenerative diseases such as amyotrophic lateral sclerosis are promising to clarify the interplay between the central nervous and immune systems. The understanding of the histological architecture providing the infrastructure of such neuro-immune interplay is also essential. This Frontiers research topic brings together fourteen articles and aims to create a platform for researchers in the field of psychoneuroimmunology to share the recent theories, hypotheses and future perspectives regarding open questions on the mechanisms of cell-cell interactions with chemical mediators among the nervous, immune and endocrine systems. We hope that this platform would reveal the relevance of the studies on multisystem interactions to enhance the understanding of the mechanisms underlying a wide variety of neurological and psychiatric disorders

Excitability of Rat Superficial Dorsal Horn Neurons Following a Neonatal Immune Challenge

Previous studies have shown that neonatal exposure to a mild inflammatory challenge, such as lipopolysaccharide (LPS, Salmonella enteriditis) results in altered pain behaviors later in life. To further characterize the impact of a neonatal immune challenge on pain processing, we examined the excitability of superficial dorsal horn (SDH) neurons following neonatal LPS exposure and subsequent responses to noxious stimulation at three time-points during early postnatal development. Wistar rats were injected with LPS (0.05 mg/kg i.p.) or saline on postnatal days (PNDs) 3 and 5, and later subjected to the formalin test at PNDs 7, 13, and 22. One hour after formalin injection into the plantar hindpaw, animals were euthanized (Ketamine, 100 mg/kg i.p.) and transverse slices from the lumbosacral spinal cord were prepared. Whole-cell patch-clamp recordings were made from SDH neurons (KCH3SO4-based internal, 22–24°C) on the ipsi- and contralateral sides of the spinal cord. Depolarising current steps were injected into SDH neurons to categorize action potential (AP) discharge. In both saline- and LPS-treated rats we observed age-related increases the percentage of neurons exhibiting tonic-firing, with concurrent decreases in single-spiking, between PND 7 and 22. In contrast, neonatal exposure to LPS failed to alter the proportions of AP discharge patterns at any age examined. We also assessed the subthreshold currents that determine AP discharge in SDH neurons. The rapid outward potassium current, IAr decreased in prevalence with age, but was susceptible to neonatal LPS exposure. Peak IAr current amplitude was greater in ipsilateral vs. contralateral SDH neurons from LPS-treated rats. Spontaneous excitatory synaptic currents (sEPSCs) were recorded to assess network excitability. Age-related increases were observed in sEPSC frequency and time course, but not peak amplitude, in both saline- and LPS-treated rats. Furthermore, sEPSC frequency was higher in ipsilateral vs. contralateral SDH neurons in LPS-treated animals. Taken together, these data suggest a neonatal immune challenge does not markedly affect the intrinsic properties of SDH neurons, however, it can increase the excitability of local spinal cord networks via altering the properties of rapid A-type currents and excitatory synaptic connections. These changes, made in neurons within spinal cord pain circuits, have the capacity to alter nociceptive signaling in the ascending pain pathway

Altered formalin-induced pain and Fos induction in the periaqueductal grey of preadolescent rats following neonatal LPS exposure

Animal and human studies have demonstrated that early pain experiences can produce alterations in the nociceptive systems later in life including increased sensitivity to mechanical, thermal, and chemical stimuli. However, less is known about the impact of neonatal immune challenge on future responses to noxious stimuli and the reactivity of neural substrates involved in analgesia. Here we demonstrate that rats exposed to Lipopolysaccharide (LPS; 0.05 mg/kg IP, Salmonella enteritidis) during postnatal day (PND) 3 and 5 displayed enhanced formalin-induced flinching but not licking following formalin injection at PND 22. This LPS-induced hyperalgesia was accompanied by distinct recruitment of supra-spinal regions involved in analgesia as indicated by significantly attenuated Fos-protein induction in the rostral dorsal periaqueductal grey (DPAG) as well as rostral and caudal axes of the ventrolateral PAG (VLPAG). Formalin injections were associated with increased Fos-protein labelling in lateral habenula (LHb) as compared to medial habenula (MHb), however the intensity of this labelling did not differ as a result of neonatal immune challenge. These data highlight the importance of neonatal immune priming in programming inflammatory pain sensitivity later in development and highlight the PAG as a possible mediator of this proces

Altered nociceptive, endocrine, and dorsal horn neuron responses in rats following a neonatal immune challenge

Summary The neonatal period is characterized by significant plasticity where the immune, endocrine, and nociceptive systems undergo fine-tuning and maturation. Painful experiences during this period can result in long-term alterations in the neurocircuitry underlying nociception, including increased sensitivity to mechanical or thermal stimuli. Less is known about the impact of neonatal exposure to mild inflammatory stimuli, such as lipopolysaccharide (LPS), on subsequent inflammatory pain responses. Here we examine the impact of neonatal LPS exposure on inflammatory pain sensitivity and HPA axis activity during the first three postnatal weeks. Wistar rats were injected with LPS (0.05 mg/kg IP, Salmonella enteritidis) or saline on postnatal days (PNDs) 3 and 5 and later subjected to the formalin test at PNDs 7, 13, and 22. One hour after formalin injection, blood was collected to assess corticosterone responses. Transverse spinal cord slices were also prepared for whole-cell patch clamp recording from lumbar superficial dorsal horn neurons (SDH). Brains were obtained at PND 22 and the hypothalamus was isolated to measure glucocorticoid (GR) and mineralocorticoid receptor (MR) transcript expression using qRT-PCR. Behavioural analyses indicate that at PND 7, no significant differences were observed between saline- or LPS-challenged rats. At PND 13, LPS-challenged rats exhibited enhanced licking (p < .01), and at PND 22, increased flinching in response to formalin injection (p < .05). LPS-challenged rats also displayed increased plasma corticosterone at PND 7 and PND 22 (p < .001) but not at PND 13 following formalin administration. Furthermore, at PND 22 neonatal LPS exposure induced decreased levels of GR mRNA and increased levels of MR mRNA in the hypothalamus. The intrinsic properties of SDH neurons were similar at PND 7 and PND 13. However, at PND 22, ipsilateral SDH neurons in LPS-challenged rats had a lower input resistance compared to their saline-challenged counterparts (p < .05). These data suggest neonatal LPS exposure produces developmentally regulated changes in formalin-induced behavioural responses, corticosterone levels, and dorsal horn neuron properties following noxious stimulation later in life. These findings highlight the importance of immune activation during the neonatal period in shaping pain sensitivity later in life. This programming involves both spinal cord neurons and the HPA axis

Time course of licking responses in PND22 male (A) and female (B) rats in response to an injection of 0.9%, 1%, and 1.1% formalin into the plantar surface of the left hindpaw.

<p>Data are presented as mean +/− SE. * <i>p</i><.05; **<i>p</i><.01, against other groups at the same time point.</p