Towards a central origin of nociceptive hypersensitivity in adult rats after a neonatal maternal separation.

Early life adversities influence a nervous system still in development with long-term consequences for later life. These include nociceptive circuit alterations critical to shape an adaptive pain response to protect the organism from potential damage. Adult rats with a history of neonatal maternal separation (NMS) display visceral and somatic nociceptive hypersensitivity and inefficient analgesic responses to stress. In this study, we have characterized the consequences of NMS on wide dynamic range neurons (WDR) in the spinal cord of anaesthetized adult rats during the nociceptive processing of hot and cold noxious information. We found that WDR neurons of NMS rats display an excessive coding of mechanical and thermal information applied at the rat's hindpaws. This nicely explains the hypernociceptive behaviours seen after noxious mechanical, cold and hot peripheral stimulation. A peripheral change in the expression of molecular transducers for these stimuli (i.e., TRPV1, TRPM8 and TRPA1) does not seem to account for this general hyperexcitability. Instead, a decreased chloride-mediated inhibitory tone on WDR neurons may play a role as indicated by the abnormal elevation of the type 1 Na-K-Cl cotransporter transcripts. Altogether, we propose that long-term consequences of NMS are associated with reduced spinal cord inhibition favouring the expression of pain hypersensitivity. We cannot exclude that this phenomenon is also present at supraspinal sites, as other NMS-associated symptoms include excessive anxiety and impaired sociability.

Overexpression of chloride importer NKCC1 contributes to the sensory-affective and sociability phenotype of rats following neonatal maternal separation.

BACKGROUND: Early life stress is known to affect the development of the nervous system and its function at a later age. It increases the risk to develop psychiatric disorders as well as chronic pain and its associated affective comorbidities across the lifespan. GABAergic inhibition is important for the regulation of central function and related behaviors, including nociception, anxiety or social interactions, and requires low intracellular chloride levels. Of particular interest, the oxytocinergic (OTergic) system exerts potent anxiolytic, analgesic and pro-social properties and is known to be involved in the regulation of chloride homeostasis and to be impaired following early life stress. METHODS: We used behavioral measures to evaluate anxiety, social interactions and pain responses in a rat model of neonatal maternal separation (NMS). Using quantitative PCR, we investigated whether NMS was associated with alterations in the expression of chloride transporters in the cerebrum and spinal cord. Finally, we evaluated the contribution of OTergic signaling and neuro-inflammatory processes in the observed phenotype. RESULTS: NMS animals displayed a long-lasting upregulation of chloride importer Na-K-Cl cotransporter type 1 (NKCC1) expression in the cerebrum and spinal cord. Neonatal administration of the NKCC1 inhibitor bumetanide or oxytocin successfully normalized the anxiety-like symptoms and the lack of social preference observed in NMS animals. Phenotypic alterations were associated with a pro-inflammatory state which could contribute to NKCC1 upregulation. CONCLUSIONS: This work suggests that an impaired chloride homeostasis, linked to oxytocin signaling dysfunction and to neuro-inflammatory processes, could contribute to the sensori-affective phenotype following NMS.

A Dual Noradrenergic Mechanism for the Relief of Neuropathic Allodynia by the Antidepressant Drugs Duloxetine and Amitriptyline.

In addition to treating depression, antidepressant drugs are also a first-line treatment for neuropathic pain, which is pain secondary to lesion or pathology of the nervous system. Despite the widespread use of these drugs, the mechanism underlying their therapeutic action in this pain context remains partly elusive. The present study combined data collected in male and female mice from a model of neuropathic pain and data from the clinical setting to understand how antidepressant drugs act. We show two distinct mechanisms by which the selective inhibitor of serotonin and noradrenaline reuptake duloxetine and the tricyclic antidepressant amitriptyline relieve neuropathic allodynia. One of these mechanisms is acute, central, and requires descending noradrenergic inhibitory controls and α2A adrenoceptors, as well as the mu and delta opioid receptors. The second mechanism is delayed, peripheral, and requires noradrenaline from peripheral sympathetic endings and ß2 adrenoceptors, as well as the delta opioid receptors. We then conducted a transcriptomic analysis in dorsal root ganglia, which suggested that the peripheral component of duloxetine action involves the inhibition of neuroimmune mechanisms accompanying nerve injury, including the downregulation of the TNF-α-NF-κB signaling pathway. Accordingly, immunotherapies against either TNF-α or Toll-like receptor 2 (TLR2) provided allodynia relief. We also compared duloxetine plasma levels in the animal model and in patients and we observed that patients' drug concentrations were compatible with those measured in animals under chronic treatment involving the peripheral mechanism. Our study highlights a peripheral neuroimmune component of antidepressant drugs that is relevant to their delayed therapeutic action against neuropathic pain.SIGNIFICANCE STATEMENT In addition to treating depression, antidepressant drugs are also a first-line treatment for neuropathic pain, which is pain secondary to lesion or pathology of the nervous system. However, the mechanism by which antidepressant drugs can relieve neuropathic pain remained in part elusive. Indeed, preclinical studies led to contradictions concerning the anatomical and molecular substrates of this action. In the present work, we overcame these apparent contradictions by highlighting the existence of two independent mechanisms. One is rapid and centrally mediated by descending controls from the brain to the spinal cord and the other is delayed, peripheral, and relies on the anti-neuroimmune action of chronic antidepressant treatment.

Peripheral and central alterations affecting spinal nociceptive processing and pain at adulthood in rats exposed to neonatal maternal deprivation.

The nociceptive system of rodents is not fully developed and functional at birth. Specifically, C fibers transmitting peripheral nociceptive information establish synaptic connections in the spinal cord already during the embryonic period that only become fully functional after birth. Here, we studied the consequences of neonatal maternal deprivation (NMD, 3 h/day, P2-P12) on the functional establishment of C fiber-mediated neurotransmission in spinal cord and of pain-related behavior. In vivo recording revealed that C fiber-mediated excitation of spinal cord neurons could be observed at P14 only in control but not in NMD rats. NMD was associated with a strong alteration in the expression of growth factors controlling C nociceptor maturation as well as two-pore domain K+ channels known to set nociceptive thresholds. In good agreement, C-type sensory neurons from NMD animals appeared to be hypoexcitable but functionally connected to spinal neurons, especially those expressing TRPV1 receptors. In vivo and in vitro recordings of lamina II spinal neurons at P14 revealed that the NMD-related lack of C fiber-evoked responses resulted from an inhibitory barrage in the spinal cord dorsal horn. Eventually, C-type sensory-spinal processing could be recovered after a delay of about 10 days in NMD animals. However, animals remained hypersensitive to noxious stimulus up to P100 and this might be due to an excessive expression of Nav1.8 transcripts in DRG neurons. Together, our data provide evidence for a deleterious impact of perinatal stress exposure on the maturation of the sensory-spinal nociceptive system that may contribute to the nociceptive hypersensitivity in early adulthood.

Neuropeptides shaping the central nervous system development: Spatiotemporal actions of VIP and PACAP through complementary signaling pathways.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are neuropeptides with wide, complementary, and overlapping distributions in the central and peripheral nervous systems, where they exert important regulatory roles in many physiological processes. VIP and PACAP display a large range of biological cellular targets and functions in the adult nervous system including regulation of neurotransmission and neuroendocrine secretion and neuroprotective and neuroimmune responses. As the main focus of the present review, VIP and PACAP also have been long implicated in nervous system development and maturation through their interaction with the seven transmembrane domain G protein-coupled receptors, PAC1, VPAC1, and VPAC2, initiating multiple signaling pathways. Compared with PAC1, which solely binds PACAP with very high affinity, VPACs exhibit high affinities for both VIP and PACAP but differ from each other because of their pharmacological profile for both natural accessory peptides and synthetic or chimeric molecules, with agonistic and antagonistic properties. Complementary to initial pharmacological studies, transgenic animals lacking these neuropeptides or their receptors have been used to further characterize the neuroanatomical, electrophysiological, and behavioral roles of PACAP and VIP in the developing central nervous system. In this review, we recapitulate the critical steps and processes guiding/driving neurodevelopment in vertebrates and superimposing the potential contribution of PACAP and VIP receptors on the given timeline. We also describe how alterations in VIP/PACAP signaling may contribute to both (neuro)developmental and adult pathologies and suggest that tuning of VIP/PACAP signaling in a spatiotemporal manner may represent a novel avenue for preventive therapies of neurological and psychiatric disorders. © 2016 Wiley Periodicals, Inc.

Behavioral and pharmacological characterization of planarian nociception.

Introduction: Pain mostly arises because specialized cells called nociceptors detect harmful or potentially harmful stimuli. In lower animals with less convoluted nervous system, these responses are believed to be purely nociceptive. Amongst invertebrate animal models, planarians are becoming popular in a wide range of pharmacological and behavioral studies beyond the field of regeneration. Recent publications led the way on pain studies by focusing on nociceptive behaviors such as the 'scrunching' gait displayed under various noxious stimuli, as opposed to the 'gliding' gait planarians usually adopt in normal conditions. Methods: In this study, we adapted commonly used nociceptive tests to further explore nociception in planarians of the species Girardia dorotocephala. By using behavioral analysis in open fields and place preferences, we managed to set up chemical, thermal and mechanical nociceptive tests. We also adapted RNA interference protocols and explored the effects of knocking down TRPA1 ion channels, one of the main effectors of chemically and thermally-induced nociceptive responses in vertebrates. Results: Consequently, we demonstrated the reliability of the scrunching gait in this planarian species, which they displayed in a dose-dependent manner when exposed to the irritant AITC. We also showed that suppressing the expression of TRPA1 ion channels completely suppressed the scrunching gait, demonstrating the involvement of TRPA1 nociceptors in this nociceptive reaction. Besides, we also explored the effects of two common analgesics that both displayed strong antinociceptive properties. First, morphine reduced the chemically-induced nociceptive scrunching gaits by more than 20% and shifted the EC50 of the dose-response curve by approximately 10 µM. Secondly, the NSAID meloxicam drastically reduced chemically-induced scrunching by up to 60% and reduced heat avoidance in place preference tests. Discussion: Thus, we managed to characterize both behavioral and pharmacological aspects of G. dorotocephala's nociception, further developing the use of planarians as a replacement model in pain studies and more globally the study of invertebrate nociception.

Effect of an intraoperative periradicular application of platelet-rich fibrin (PRF) on residual post-surgical neuropathic pain after disc herniation surgery: study protocol for NeuroPRF, a randomized controlled trial.

BACKGROUND: The prevalence of post-surgical lumbar neuropathic radiculopathy is approximately 30%. Poor response to the recommended treatments for neuropathic pain, namely antidepressants and/or gabapentinoids, requires the development of new techniques to prevent chronic pain. One such well-tolerated technique is the administration of autologous plasma enriched in platelets and fibrin (PRF). This approach is largely used in regenerative medicine owing to the anti-inflammatory and analgesic properties of PRF. It could also be an interesting adjuvant to surgery, as it reduces neurogenic inflammation and promotes nerve recovery, thereby reducing the incidence of residual postoperative chronic pain. The aim of the present study is to evaluate the benefit of periradicular intraoperative application of PRF on the residual postsurgical neuropathic pain after disc herniation surgery. METHODS: A randomized, prospective, interventional, controlled, single-blind study with evaluation by a blind outcome assessor will be performed in Strasbourg University Hospital. We will compare a control group undergoing conventional surgery to an experimental group undergoing surgery and periradicular administration of PRF (30 patients in each arm). The primary outcome is the intensity of postoperative neuropathic radicular pain, measured by a visual analog scale (VAS) at 6 months post-surgery. The secondary outcomes are the characteristics of neuropathic pain (NPSI), the quality of life (SF-12 and PGIC), the presence of anxiety/depression symptoms (HAD), and the consumption of analgesics. We will also carry out transcriptomic analysis of a panel of pro- and anti-inflammatory cytokines in blood samples, before surgery and at 6 months follow-up. These gene expression results will be correlated with clinical data, in particular, with the apparition of postoperative neuropathic pain. DISCUSSION: This study is the first randomized controlled trial to assess the efficacy of PRF in the prevention of neuropathic pain following surgery for herniated disc. This study addresses not only a clinical question but will also provide information on the physiopathological mechanisms of neuropathic pain. TRIAL REGISTRATION: This study is registered at ClinicalTrials.gov: NCT05196503 , February 24, 2022.

Systemic inflammation disrupts the developmental program of white matter.

OBJECTIVE: Perinatal inflammation is a major risk factor for neurological deficits in preterm infants. Several experimental studies have shown that systemic inflammation can alter the programming of the developing brain. However, these studies do not offer detailed pathophysiological mechanisms, and they rely on relatively severe infectious or inflammatory stimuli that most likely do not reflect the levels of systemic inflammation observed in many human preterm infants. The goal of the present study was to test the hypothesis that moderate systemic inflammation is sufficient to alter white matter development. METHODS: Newborn mice received twice-daily intraperitoneal injections of interleukin-1ß (IL-1ß) over 5 days and were studied for myelination, oligodendrogenesis, and behavior and with magnetic resonance imaging (MRI). RESULTS: Mice exposed to IL-1ß had a long-lasting myelination defect that was characterized by an increased number of nonmyelinated axons. They also displayed a reduction of the diameter of the myelinated axons. In addition, IL-1ß induced a significant reduction of the density of myelinating oligodendrocytes accompanied by an increased density of oligodendrocyte progenitors, suggesting a partial blockade in the oligodendrocyte maturation process. Accordingly, IL-1ß disrupted the coordinated expression of several transcription factors known to control oligodendrocyte maturation. These cellular and molecular abnormalities were correlated with a reduced white matter fractional anisotropy on diffusion tensor imaging and with memory deficits. INTERPRETATION: Moderate perinatal systemic inflammation alters the developmental program of the white matter. This insult induces a long-lasting myelination deficit accompanied by cognitive defects and MRI abnormalities, further supporting the clinical relevance of the present data.

Planarian nociception: Lessons from a scrunching flatworm.

In addition to being studied for their exceptional regeneration abilities, planarians (i.e., flatworms) have also been extensively used in the context of pharmacological experiments during the past century. Many researchers used planarians as a model system for the study of drug abuse because they display high similarities with the nervous system of vertebrates at cellular and molecular levels (e.g., neuronal morphology, neurotransmitter ligands, and receptor function). This research field recently led to the discovery of causal relationships between the expression of Transient Receptor Potential ion channels in planarians and their behavioral responses to noxious stimuli such as heat, cold or pharmacological analogs such as TRP agonists, among others. It has also been shown that some antinociceptive drugs modulate these behaviors. However, among the few authors that tried to implement a full behavior analysis, none reached a consensual use of the terms used to describe planarian gaits yet, nor did they establish a comprehensive description of a potential planarian nociceptive system. The aim of this review is therefore to aggregate the ancient and the most recent evidence for a true nociceptive behavior in planarians. It also highlights the convenience and relevance of this invertebrate model for nociceptive tests and suggests further lines of research. In regards to past pharmacological studies, this review finally discusses the opportunities given by the model to extensively screen for novel antinociceptive drugs.

Antiallodynic action of phosphodiesterase inhibitors in a mouse model of peripheral nerve injury.

Neuropathic pain arises as a consequence of a lesion or disease affecting the somatosensory nervous system. It is accompanied by neuronal and non-neuronal alterations, including alterations in intracellular second messenger pathways. Cellular levels of 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) are regulated by phosphodiesterase (PDE) enzymes. Here, we studied the impact of PDE inhibitors (PDEi) in a mouse model of peripheral nerve injury induced by placing a cuff around the main branch of the sciatic nerve. Mechanical hypersensitivity, evaluated using von Frey filaments, was relieved by sustained treatment with the non-selective PDEi theophylline and ibudilast (AV-411), with PDE4i rolipram, etazolate and YM-976, and with PDE5i sildenafil, zaprinast and MY-5445, but not by treatments with PDE1i vinpocetine, PDE2i EHNA or PDE3i milrinone. Using pharmacological and knock-out approaches, we show a preferential implication of delta opioid receptors in the action of the PDE4i rolipram and of both mu and delta opioid receptors in the action of the PDE5i sildenafil. Calcium imaging highlighted a preferential action of rolipram on dorsal root ganglia non-neuronal cells, through PDE4B and PDE4D inhibition. Rolipram had anti-neuroimmune action, as shown by its impact on levels of the pro-inflammatory cytokine tumor necrosis factor-α (TNFα) in the dorsal root ganglia of mice with peripheral nerve injury, as well as in human peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharides. This study suggests that PDEs, especially PDE4 and 5, may be targets of interest in the treatment of neuropathic pain.

Vulnerability of white matter towards antenatal hypoxia is linked to a species-dependent regulation of glutamate receptor subunits.

White-matter damage is a leading cause of neurological handicap. Although hypoxia-ischemia and excitotoxicity are major pathogenic factors, a role for genetic influences was suggested recently. Thus, protracted gestational hypoxia was associated with white-matter damage (WMD) in rat pups but not in mouse pups. Indeed, microglial activation and vessel-wall density on postnatal days (P)1 and P10 were found increased in both mouse and rat pups, but cell death, astrogliosis, and myelination were only significantly altered in hypoxic rat pups. We investigated whether this species-related difference was ascribable to effects of antenatal hypoxia on the expression of glutamate receptor subunits by using immunocytochemistry, PCR, and excitotoxic double hit insult. Quantitative PCR in hypoxic mouse pups on P1 showed 2- to 4-fold down-regulation of the AMPA-receptor subunits -1, 2, and -4; of the kainate-receptor subunit GluR7; and of the metabotropic receptor subunits mGluR1, -2, -3, -5, and -7. None of the glutamate-receptor subunits was down-regulated in the hypoxic rat pups. NR2B was the only NMDA-receptor subunit that was down-regulated in hypoxic mice but not in hypoxic rat on P1. Ifenprodil administration to induce functional inhibition of NMDA containing NR2B-subunit receptors prevented hypoxia-induced myelination delay in rat pups. Intracerebral injection of a glutamate agonist produced a larger decrease in ibotenate-induced excitotoxic lesions in hypoxic mouse pups than in normoxic mouse pups. Gestational hypoxia may regulate the expression of specific glutamate-receptor subunits in fetal mice but not in fetal rats. Therefore, genetic factors may influence the susceptibility of rodents to WMD.

Behavioural and neuroplastic effects of the new-generation antidepressant agomelatine compared to fluoxetine in glucocorticoid receptor-impaired mice.

Major depression is associated with reduced hippocampal volume linked to stress and high glucocorticoid secretion. Glucocorticoid receptor-impaired (GR-i) mice, a transgenic model for affective disorders with hypothalamic-pituitary-adrenal (HPA) axis feedback control deficit, were used to assess the antidepressant-like effects of the mixed melatonin receptor agonist/5-HT(2C) receptor antagonist, agomelatine, compared to the selective 5-HT reuptake inhibitor (SSRI), fluoxetine, on hippocampal neurogenesis, GR and BDNF expression and antidepressant-responsive behaviour (tail suspension test, TST). GR-i and paired wild-type (WT) mice were given acute or chronic (21 d) treatment with these drugs. Both hippocampal cell proliferation and BDNF mRNA expression were down-regulated in GR-i mice, and these alterations were reversed by chronic agomelatine and fluoxetine treatments, whereas GR mRNA down-regulation was reversed only by agomelatine. Furthermore, chronic agomelatine, but not fluoxetine, increased survival of newly formed cells in the ventral part of the hippocampus without changing their phenotypic differentiation into neurons. In the TST, the enhanced immobility of GR-i mice was reduced to WT level by acute (but not chronic) fluoxetine and chronic (but not acute) agomelatine. These results indicate that agomelatine reversed the neuroplastic changes and helpless behaviour associated with HPA axis alterations in GR-i mice, suggesting neurobiological and behavioural effects mostly similar to those typically seen with classical antidepressants such as fluoxetine, but through clearly distinct mechanisms.

Systemic inflammation sensitizes the neonatal brain to excitotoxicity through a pro-/anti-inflammatory imbalance: key role of TNFalpha pathway and protection by etanercept.

Systemic inflammation sensitizes the perinatal brain to an ischemic/excitotoxic insult but the mechanisms are poorly understood. We hypothesized that the mechanisms involve an imbalance between pro- and anti-inflammatory factors. A well characterized mouse model where a systemic injection of IL-1beta during the first five postnatal days (inflammatory insult) is combined with an intracerebral injection of the glutamatergic analogue ibotenate (excitotoxic insult) at postnatal day 5 was used. Following the inflammatory insult alone, there was a transient induction of IL-1beta and TNFalpha, compared with controls measured by quantitative PCR, ELISA, and Western blot. Following the combined inflammatory and excitotoxic insult, there was an induction of IL-1beta, TNFalpha, and IL-6 but not of IL-10 and TNFR1, indicating an altered pro-/anti-inflammatory balance after IL-1beta sensitized lesion. We then tested the hypothesis that the TNFalpha pathway plays a key role in the sensitization and insult using TNFalpha blockade (etanercept) and TNFalpha(-/-) mice. Etanercept given before the insult did not affect brain damage, but genetic deletion of TNFalpha or TNFalpha blockade by etanercept given after the combined inflammatory and excitotoxic insult reduced brain damage by 50%. We suggest this protective effect was centrally mediated, since systemic TNFalpha administration in the presence of an intact blood-brain barrier did not aggravate the damage and etanercept almost abolished cerebral TNFalpha production. In summary, sensitization was, at least partly, mediated by an imbalance between pro- and anti-inflammatory cytokines. Cerebral TNFalpha played a key role in mediating brain damage after the combined inflammatory and excitatory insult.

Dynamics of somatostatin type 2A receptor cargoes in living hippocampal neurons.

Despite the large number of G-protein-coupled receptor (GPCR) types expressed in the CNS, little is known about their dynamics in neuronal cells. Dynamic properties of the somatostatin type 2A receptor were therefore examined in resting conditions and after agonist activation in living hippocampal neurons. Using fluorescence recovery after photobleaching experiments, we found that, in absence of ligand, the sst(2A) receptor is mobile and laterally and rapidly diffuse in neuronal membranes. We then observed by live-cell imaging that, after agonist activation, membrane-associated receptors induce the recruitment of beta-arrestin 1-enhanced green fluorescent protein (EGFP) and beta-arrestin 2-EGFP to the plasma membrane. In addition, beta-arrestin 1-EGFP translocate to the nucleus, suggesting that this protein could serve as a nuclear messenger for the sst(2A) receptor in neurons. Receptors are then recruited to preexisting clathrin coated pits, form clusters that internalize, fuse, and move to a perinuclear compartment that we identified as the trans-Golgi network (TGN), and recycle. Receptor cargoes are transported through a microtubule-dependent process directly from early endosomes/recycling endosomes to the TGN, bypassing the late endosomal compartment. Together, these results provide a comprehensive description of GPCR trafficking in living neurons and provide compelling evidence that GPCR cargoes can recycle through the TGN after endocytosis, a phenomenon that has not been anticipated from studies of non-neuronal cells.

Disruption of the PACAP gene promotes medulloblastoma in ptc1 mutant mice.

Hedgehog (Hh) proteins and cAMP-dependent protein kinase A (PKA) generally play opposing roles in developmental patterning events. Humans and mice heterozygous for mutations in the sonic hedgehog (Shh) receptor gene patched-1 (ptc1) have an increased incidence of certain types of cancer, including medulloblastoma (MB), a highly aggressive tumor of the cerebellum. Despite the importance of PKA in Hh signaling, little is known about how PKA activity is regulated in the context of Hh signaling, or the consequences of improper regulation. One molecule that can influence PKA activity is pituitary adenylyl cyclase-activating peptide (PACAP), which has been shown to regulate cerebellar granule precursor proliferation in vitro, a cell population thought to give rise to MB. To test for a PACAP/Hh interaction in the initiation or propagation of these tumors, we introduced a PACAP mutation into ptc1 mutant mice. Deletion of a single copy of PACAP increased MB incidence approximate 2.5-fold, to 66%, thereby demonstrating that PACAP exerts a powerful inhibitory action on the induction, growth or survival of these tumors. Tumors from PACAP/ptc1 mutant mice retained PACAP receptor gene expression, and exhibited superinduction of Hh target genes compared to those from ptc1+/- mice. Moreover, PACAP inhibited proliferation of cell lines derived from tumors in a PKA-dependent manner, and inhibited expression of the Hh target gene gli1. The results provide genetic evidence that PACAP acts as a physiological factor that regulates the pathogenesis of Hh pathway-associated MB tumors.

Ontogeny of MMPs and TIMPs in the murine neocortex.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) have been implicated in normal brain development, adult stroke, and, more recently, perinatal brain injury. Here, our objective was to obtain comprehensive and comparative data on the ontogeny of MMP-2, MMP-9, TIMP-1, and TIMP-2 in the neocortex of male and female mice belonging to various strains, from embryonic life to adulthood. We used gelatin zymography, ELISA, and real-time PCR analyses. MMP-2, MMP-9, and TIMP-1 activity and/or expression peaked during embryonic life and the early neonatal period, whereas TIMP-2 peaked during the first two postnatal weeks. Comparable results were obtained in all the mouse strains except BALB/c, where MMP-2 levels were considerably lower at all ages compared with the other strains. No gender effect was observed on any of the study parameters. This comprehensive study will serve as a basis for future investigations into the role for MMPs and TIMPs in normal brain development and prenatal brain injury.

Cholecalciferol (Vitamin D3) Reduces Rat Neuropathic Pain by Modulating Opioid Signaling.

The impact of vitamin D on sensory function, including pain processing, has been receiving increasing attention. Indeed, vitamin D deficiency is associated with various chronic pain conditions, and several lines of evidence indicate that vitamin D supplementation may trigger pain relief. However, the underlying mechanisms of action remain poorly understood. We used inflammatory and non-inflammatory rat models of chronic pain to evaluate the benefits of vitamin D3 (cholecalciferol) on pain symptoms. We found that cholecalciferol supplementation improved mechanical nociceptive thresholds in monoarthritic animals and reduced mechanical hyperalgesia and cold allodynia in a model of mononeuropathy. Transcriptomic analysis of cerebrum, dorsal root ganglia, and spinal cord tissues indicate that cholecalciferol supplementation induces a massive gene dysregulation which, in the cerebrum, is associated with opioid signaling (23 genes), nociception (14), and allodynia (8), and, in the dorsal root ganglia, with axonal guidance (37 genes) and nociception (17). Among the identified cerebral dysregulated nociception-, allodynia-, and opioid-associated genes, 21 can be associated with vitamin D metabolism. However, it appears that their expression is modulated by intermediate regulators such as diverse protein kinases and not, as expected, by the vitamin D receptor. Overall, several genes-Oxt, Pdyn, Penk, Pomc, Pth, Tac1, and Tgfb1-encoding for peptides/hormones stand out as top candidates to explain the therapeutic benefit of vitamin D3 supplementation. Further studies are now warranted to detail the precise mechanisms of action but also the most favorable doses and time windows for pain relief.

Loss of Cubilin, the intrinsic factor-vitamin B12 receptor, impairs visceral endoderm endocytosis and endodermal patterning in the mouse.

The visceral endoderm is a polarized epithelial monolayer necessary for early embryonic development in rodents. A key feature of this epithelium is an active endocytosis and degradation of maternal nutrients, in addition to being the source of various signaling molecules or inhibitors required for the differentiation and patterning of adjacent embryonic tissues. Endocytosis across the visceral endoderm epithelium involves specific cell surface receptors and an extensive sub-membrane vesicular system with numerous apical vacuoles/lysosomes. We previously reported that Cubilin, the endocytic receptor for intrinsic factor-vitamin B12, albumin and apolipoproteinA-I/HDL allows maternal nutrient uptake by the visceral endoderm. In the present study, we show that the germline ablation of Cubilin impairs endodermal and mesodermal patterning, and results in developmental arrest at gastrulation. Notably, visceral endoderm dispersal is impeded in Cubilin null embryos. We further confirm the essential role of Cubilin in nutrient internalization by the early visceral endoderm and highlight its involvement in the formation of apical vacuoles. Our results reveal essential roles for Cubilin in early embryonic development, and suggest that in addition to its nutritive function, Cubilin sustains signaling pathways involved in embryonic differentiation and patterning.

Chronic mild stress during gestation worsens neonatal brain lesions in mice.

Cerebral palsy remains a public health priority. Recognition of factors of susceptibility to perinatal brain lesions is key for the prevention of cerebral palsy. In most cases, the pathophysiology of these lesions is thought to involve prior exposure to predisposing factors that make the developing brain more vulnerable to perinatal events. The present study tested the hypothesis that exposure to chronic minimal stress throughout gestation would sensitize the offspring to neonatal excitotoxic brain lesions, which mimic lesions observed in cerebral palsy. Pregnant mice were exposed to chronic, ultramild stress, applied throughout gestation. Neonatal brain lesions were induced by intracerebral injection of glutamate analogs. Excitotoxic lesions were significantly worsened in pups exposed to gestational stress. Stress induced a significant rise of circulating corticosterone levels both in pregnant mothers and in newborn pups. The deleterious effects of stress on excitotoxicity were totally suppressed in mice with reduced levels of glucocorticoid receptors. Stress induced a significant increase of neopallial NMDA binding sites in the offspring. At adulthood, animals exposed to stress and neonatal excitotoxic challenge showed a significant impairment in the Morris water maze test when compared with animals exposed to the excitotoxic challenge but not the gestational stress. These findings suggest that stress during gestation, which may mimic low-level stress in human pregnancy, could be a novel risk factor for cerebral palsy.

Potential role of glucocorticoid signaling in the formation of pancreatic islets in the human fetus.

Glucocorticoids have been suggested to play a role in programming late adult disorders like diabetes during fetal life. Recent work in rodents showed their role in pancreas development by modulating the expression of transcription factors. The aim of this work was to investigate their possible implication in human pancreas development. The ontogenesis of glucocorticoid receptor (GR) and several pancreatic transcription factors was studied by immunohistochemistry and RT-PCR on human fetal pancreatic specimens. At 6 wk of development (wd) insulin promoting factor 1 (IPF1) was expressed in the majority of epithelial cells forming tubular structures while GR was present in the mesenchyme, suggesting an early role of glucocorticoids, before endocrine and exocrine differentiation. Only GR alpha (active form) mRNA was expressed from 6 wk onwards while GR beta (inactive form) was never observed. The first insulin cells did not express IPF1 or GR. Islet formation occurred from 10 wd as IPF1-positive cells started to express simultaneously insulin and GR. This coexpression in beta cells persisted until adulthood. The mRNA expression profiles confirmed immunohistochemistry and showed the early expression of crucial transcription factors. In conclusion, the presence of the active GR isoform around islet formation supports the novel idea that glucocorticoids could modulate human pancreas development.