Opioid withdrawal behavior in spiny mice: A novel preclinical model of neonatal opioid withdrawal syndrome (NOWS).

As the opioid epidemic continues to grow, opioid use among pregnant women is increasing significantly. This has led to a steady rise in the number of infants born with neonatal opioid withdrawal syndrome (NOWS). Although short-term withdrawal symptoms associated with NOWS are well characterized, there are many gaps in our understanding of the short and long-term effects of prenatal opioid exposure. Current animal models of NOWS are limited by shortened gestational periods, large litter sizes, and primary organogenesis occurring after birth. This often leads to postnatal treatment to mimic drug exposure during third-trimester development. Using the unique rodent species Acomys cahirinus, more commonly known as spiny mice, which have an extended 40-day gestation period, small litter sizes, and increased in utero organogenesis we aim to study the short-term effects of prenatal morphine exposure by assessing withdrawal behavior. To model maternal opioid use, dams were treated daily with morphine (10 and 30 mg/kg S.C.) beginning on gestation day 19 until the day of birth; this resulted in a cumulative exposure of 19-21 days. Withdrawal behaviors for each pup were recorded daily between postnatal days 0-7 (PND 0-7). Our study found that prenatal morphine exposure in spiny mice led to an increase in withdrawal behavior throughout the early postnatal period and validated the use of this species as a novel pre-clinical model of NOWS. We are hopeful this rodent model will further our understanding of the short and long-term consequences of prenatal opioid exposure on neurodevelopment and behavior.

Genetic Deletion of PGF2α-FP Receptor Exacerbates Brain Injury Following Experimental Intracerebral Hemorrhage.

Background: The release of inflammatory molecules such as prostaglandins (e.g., PGF2α) is associated with brain damage following an intracerebral hemorrhagic (ICH) stroke; however, the role of PGF2α and its cognate FP receptor in ICH remains unclear. This study focused on investigating the role of the FP receptor as a target for novel neuroprotective drugs in a preclinical model of ICH, aiming to investigate the contribution of the PGF2α-FP axis in modulating functional recovery and anatomical outcomes following ICH. Results: Neurological deficit scores in FP-/- mice were significantly higher compared to WT mice 72 h after ICH (6.1 ± 0.7 vs. 3.1 ± 0.8; P < 0.05). Assessing motor skills, the total time mice stayed on the rotating rod was significantly less in FP-/-mice compared to WT mice 24 h after ICH (27.0 ± 7.5 vs. 52.4 ± 11.2 s; P < 0.05). Using grip strength to quantify forepaw strength, results showed that the FP-/- mice had significantly less strength compared to WT mice 72 h after ICH (96.4 ± 17.0 vs. 129.6 ± 5.9 g; P < 0.01). In addition to the behavioral outcomes, histopathological measurements were made. In Cresyl violet stained brain sections, the FP-/- mice showed a significantly larger lesion volume compared to the WT (15.0 ± 2.2 vs. 3.2 ± 1.7 mm3; P < 0.05 mice.) To estimate the presence of ferric iron in the peri-hematoma area, Perls' staining was performed, which revealed that FP-/- mice had significantly greater staining than the WT mice (186.3 ± 34.4% vs. 86.9 ± 13.0% total positive pixel counts, P < 0.05). Immunoreactivity experiments on brain sections from FP-/- and WT mice post-ICH were performed to monitor changes in microgliosis and astrogliosis using antibodies against Iba1 and GFAP respectively. These experiments showed that FP-/- mice had a trend toward greater astrogliosis than WT mice post-ICH. Conclusion: We showed that deletion of the PGF2α FP receptor exacerbates behavioral impairments and increases lesion volumes following ICH compared to WT-matched controls.Detailed mechanisms responsible for these novel results are actively being pursued.

Gender Differences in Withdrawing Infants.

Objective. To assess gender differences in infants diagnosed with neonatal abstinence syndrome at the Cabell Huntington Hospital in Huntington, West Virginia. Methods. This is a single-site retrospective chart review involving 97 infants born treated for neonatal abstinence syndrome at the Cabell Huntington Hospital between April and December 2015. Data were obtained from electronic medical records using a secure online survey tool designed using Qualtrics. Maternal demographics and drug screenings were collected. Infant information was collected for the first 7 days of life including withdrawal symptoms, treatment, and growth parameters. These data were analyzed based on gender, male (N = 62) and female (N = 35), to assess any gender differences among the infants. Results. No significant differences were found regarding birth weight, length, and gestational age between male and female infants. Differences among the percentage of symptoms experienced were found with females experiencing a greater percentage of symptoms affecting the autonomic nervous system compared with males. Significant differences in head circumference were found in these infants; females were found to have a greater head circumference at time of birth compared with males (P = .003), whereas at time of discharge head circumference was greater in males than in females (P = .035). Conclusion. Differences in symptoms, physical characteristics, and methadone treatment were found between male and female infants diagnosed with neonatal abstinence syndrome at the Cabell Huntington Hospital during 2015. Further studies are needed to assess both the short- and long-term effects of antenatal drug abuse.

Neuroprotective role of prostaglandin PGE2 EP2 receptor in hemin-mediated toxicity.

Heme (Fe(2+) protoporphyrin IX) and hemin (Fe(3+)), the prosthetic group of hemoprotein, are cytotoxic due to their ability to contribute to the production of reactive oxygen species, increased intracellular calcium levels, and stimulate glutamate-mediated excitotoxicity. Previous work by our group showed that blockade of the prostaglandin E2 (PGE2)-EP1 receptor reduced hemin-induced cytotoxicity in primary cortical neuronal cultures. However, the role of the prostaglandin E2 (PGE2)-EP2 receptor in hemin neurotoxicity remains unclear. Activation of the EP2 receptor in neurons results in increased cyclic AMP (cAMP) and protein kinase A signaling; therefore, we hypothesized that the activation of the EP2 receptor decreases hemin neurotoxicity. Using postnatal primary cortical neurons cultured from wildtype-control (WT) and EP2(-/-) mice, we investigated the role of the EP2 receptor in hemin neurotoxicity by monitoring cell survival with the Calcein-AM live-cell and lactate dehydrogenase assays. MitoTracker staining was also performed to determine how mitochondria were affected by hemin. Hemin neurotoxicity in EP2(-/-) neurons was 37.2 ± 17.0% greater compared to WT neurons. Of interest, cotreatment with the EP2 receptor agonist, butaprost (1 and 10 µM), significantly attenuated hemin neurotoxicity by 55.7 ± 21.1% and 60.1 ± 14.8%, respectively. To further investigate signaling mechanisms related to EP2 receptor mediating cytoprotection, neurons were cotreated with hemin and activators/inhibitors of both the cAMP-protein kinase A/exchange protein directly activated by cAMP (Epac) pathways. Forskolin, a cAMP activator, and 8-pCPT-cAMP, an Epac activator, both attenuated hemin neurotoxicity by 78.8 ± 22.2% and 58.4 ± 9.8%, respectively, as measured using the lactate dehydrogenase assay. Together, the results reveal that activation of the EP2 receptor is protective against hemin neurotoxicity in vitro and these findings suggest that neuroprotection occurs through the cAMP-Epac pathway in neuronal cultures. Therefore, activation of the EP2 receptor could be used to minimize neuronal damage following exposure to supraphysiological levels of hemin.

Role of vasopressin and its antagonism in stroke related edema.

Although many approaches have been tried in the attempt to reduce the devastating impact of stroke, tissue plasminogen activator for thromboembolic stroke is the only proved, effective acute stroke treatment to date. Vasopressin, an acute-phase reactant, is released after brain injury and is partially responsible for the subsequent inflammatory response via activation of divergent pathways. Recently there has been increasing interest in vasopressin because it is implicated in inflammation, cerebral edema, increased intracerebral pressure, and cerebral ion and neurotransmitter dysfunctions after cerebral ischemia. Additionally, copeptin, a byproduct of vasopressin production, may serve as a promising independent marker of tissue damage and prognosis after stroke, thereby corroborating the role of vasopressin in acute brain injury. Thus, vasopressin antagonists have a potential role in early stroke intervention, an effect thought to be mediated via interactions with aquaporin receptors, specifically aquaporin-4. Despite some ambiguity, vasopressin V1a receptor antagonism has been consistently associated with attenuated secondary brain injury and edema in experimental stroke models. The role of the vasopressin V2 receptor remains unclear, but perhaps it is involved in a positive feedback loop for vasopressin expression. Despite the encouraging initial findings we report here, future research is required to characterize further the utility of vasopressin antagonists in treatment of stroke.

Contribution of PGE2 EP1 receptor in hemin-induced neurotoxicity.

Although hemin-mediated neurotoxicity has been linked to the production of free radicals and glutamate excitotoxicity, the role of the prostaglandin E2 (PGE2)-EP1 receptor remains unclear. Activation of the EP1 receptor in neurons results in increased intracellular calcium levels; therefore, we hypothesize that the blockade of the EP1 receptor reduces hemin neurotoxicity. Using postnatal primary cortical neurons cultured from wild-type (WT) and EP1(-/-) mice, we investigated the EP1 receptor role in hemin neurotoxicity measured by lactate dehydrogenase (LDH) cell survival assay. Hemin (75 µM) induced greater release of LDH in WT (34.7 ± 4.5%) than in EP1(-/-) (27.6 ± 3.3%) neurons. In the presence of the EP1 receptor antagonist SC-51089, the hemin-induced release of LDH decreased. To further investigate potential mechanisms of action, we measured changes in the intracellular calcium level [Ca(2+)]i following treatment with 17-phenyl trinor PGE2 (17-pt-PGE2) a selective EP1 agonist. In the WT neurons, 17-pt-PGE2 dose-dependently increased [Ca(2+)]i. However, in EP1(-/-) neurons, [Ca(2+)]i was significantly attenuated. We also revealed that hemin dose-dependently increased [Ca(2+)]i in WT neurons, with a significant decrease in EP1(-/-) neurons. Both 17-pt-PGE2 and hemin-induced [Ca(2+)]i were abolished by N-methyl-D-aspartic (NMDA) acid receptor and ryanodine receptor blockers. These results suggest that blockade of the EP1 receptor may be protective against hemin neurotoxicity in vitro. We speculate that the mechanism of hemin neuronal death involves [Ca(2+)]i mediated by NMDA acid receptor-mediated extracellular Ca(2+) influx and EP1 receptor-mediated intracellular release from ryanodine receptor-operated Ca(2+) stores. Therefore, blockade of the EP1 receptor could be used to minimize neuronal damage following exposure to supraphysiological levels of hemin.

Putative role of prostaglandin receptor in intracerebral hemorrhage.

Each year, approximately 795,000 people experience a new or recurrent stroke. Of all strokes, 84% are ischemic, 13% are intracerebral hemorrhage (ICH) strokes, and 3% are subarachnoid hemorrhage strokes. Despite the decreased incidence of ischemic stroke, there has been no change in the incidence of hemorrhagic stroke in the last decade. ICH is a devastating disease 37-38% of patients between the ages of 45 and 64 die within 30 days. In an effort to prevent ischemic and hemorrhagic strokes we and others have been studying the role of prostaglandins and their receptors. Prostaglandins are bioactive lipids derived from the metabolism of arachidonic acid. They sustain homeostatic functions and mediate pathogenic mechanisms, including the inflammatory response. Most prostaglandins are produced from specific enzymes and act upon cells via distinct G-protein coupled receptors. The presence of multiple prostaglandin receptors cross-reactivity and coupling to different signal transduction pathways allow differentiated cells to respond to prostaglandins in a unique manner. Due to the number of prostaglandin receptors, prostaglandin-dependent signaling can function either to promote neuronal survival or injury following acute excitotoxicity, hypoxia, and stress induced by ICH. To better understand the mechanisms of neuronal survival and neurotoxicity mediated by prostaglandin receptors, it is essential to understand downstream signaling. Several groups including ours have discovered unique roles for prostaglandin receptors in rodent models of ischemic stroke, excitotoxicity, and Alzheimer disease, highlighting the emerging role of prostaglandin receptor signaling in hemorrhagic stroke with a focus on cyclic-adenosine monophosphate and calcium (Ca(2+)) signaling. We review current ICH data and discuss future directions notably on prostaglandin receptors, which may lead to the development of unique therapeutic targets against hemorrhagic stroke and brain injuries alike.

Evaluation of leukemia inhibitory factor (LIF) in a rat model of postoperative pain.

UNLABELLED: Postoperative pain remains a significant problem despite optimal treatment with current pharmaceutical agents. In an effort to provide better postoperative pain control, there is a need to understand the factors that contribute to the development of pain after surgery. Leukemia inhibitory factor (LIF) is a pleiotropic cytokine released from tissues after injury. We hypothesized that LIF expression in skin, muscle, and dorsal root ganglion (DRG) would increase after plantar incision. The mRNA and protein expression of LIF and LIF receptor (LIF-R) were measured after plantar incision in the rat. Pain behaviors, immunohistochemistry, and C-fiber heat responses to LIF were also studied. LIF expression increased after incision in skin and muscle, and LIF-R was present in large and small DRG neurons. LIF administration to the hindpaw increased pain behaviors, a process that was reversed by anti-LIF. However, LIF and anti-LIF treatment at the time of incision did not augment or ameliorate pain behaviors. LIF treatment activated the second messenger system, JAK-STAT3, in cultured DRG neurons, but failed to alter spontaneous activity or heat responses in C-fiber nociceptors. In conclusion, LIF is not a target for postoperative analgesia; LIF may be important for skin and muscle repair and regeneration after incision. PERSPECTIVE: This article highlights an incision pain model for the study of factors involved in nociception. The study demonstrates that LIF in is an unlikely target for novel early postoperative analgesics.

Dual regulation of mu opioid receptors in SK-N-SH neuroblastoma cells by morphine and interleukin-1ß: evidence for opioid-immune crosstalk.

Treatment of SK-N-SH cells with morphine and interleukin-1beta (IL-1ß) produced dual regulation of the mRNA for the human mu opioid receptor (MOR) protein. Morphine produced a decrease in the MOR mRNA while IL-1ß increased it, as assessed by real-time quantitative PCR. These data were consistent with immunocytochemical studies of treated and untreated cells. Morphine-mediated down-regulation of MOR was blocked by naltrexone and IL-1ß-induced up-regulation of MOR was blocked by interleukin-1 receptor type 1 antagonist. Immune-opioid crosstalk was examined by IL-1ß and morphine co-treatment. These data are the first to show dual regulation of MOR in neuroblastoma cells.

Beta-funaltrexamine inhibits inducible nitric-oxide synthase expression in human astroglial cells.

The inducible isoform of nitric-oxide synthase (iNOS) is involved in neuropathogenesis associated with infection and disease in the brain. Hence, there is considerable interest in the identification of therapeutic interventions to prevent iNOS-mediated pathology. Astroglia are a major site of iNOS expression during neuropathogenesis. To mimic a key component of neuroinflammation, human A172 astroglial cells were exposed in vitro to a cytokine mixture containing interferon gamma, tumor necrosis factor alpha, and interleukin-1beta, resulting in significant iNOS expression. Next, we assessed the effects of the mu opioid receptor antagonist, beta-funaltrexamine (beta-FNA), on cytokine induced iNOS expression in human astroglia. beta-FNA dose-dependently inhibited iNOS expression. beta-FNA transcriptionally (or pre-transcriptionally) inhibited cytokine-induced iNOS activation as indicated by a significant decrease in NOS2 messenger RNA expression. Further characterization of the novel, anti-inflammatory actions of beta-FNA may provide insights for pharmacologic strategies to treat or prevent brain pathologies associated with neuroinflammation.

Cloning and bioinformatics of amphibian mu, delta, kappa, and nociceptin opioid receptors expressed in brain tissue: evidence for opioid receptor divergence in mammals.

Opioid agonists produce analgesia in humans and other mammals by binding to three distinct types of G protein-coupled receptors; mu (MOR), delta (DOR), and kappa (KOR) opioid receptors. A fourth member of the opioid receptor family is the nociceptin or orphanin FQ receptor (ORL), however the role of the ORL receptor in analgesia is less clear. In the Northern grass frog, Rana pipiens, systemic and central administration of morphine and selective MOR, DOR, and KOR agonists produced dose-dependent antinociceptive effects blocked by the general opioid antagonist, naltrexone. The present study reports on the sequence, expression, and bioinformatics of four opioid receptor cDNAs cloned from Rana pipiens; rpMOR, rpDOR, rpKOR, and rpORL. These were the first opioid receptors cloned from a species of Class Amphibia, are selectively expressed in brain tissue, and show 70-84% identity to their homologous mammalian opioid receptors. Comparisons within species showed that MOR, DOR, and KOR proteins are significantly less divergent in earlier-evolved vertebrates compared to humans and other mammals. Among the four types of opioid receptors, MOR proteins show the least sequence variation among the six vertebrate species. Additionally, phylogenetic analysis supports the hypothesis that the family of opioid receptor proteins are coded by four genes that arose from two gene duplications of a single ancestral opioid receptor gene.

Systemic and spinal administration of the mu opioid, remifentanil, produces antinociception in amphibians.

Remifentanil is a relatively new opioid analgesic related to the fentanyl family of mu opioid receptor agonists and is used clinically for its unique property of having an ultra-short duration of action. However, there is little preclinical data on the analgesic (antinociceptive) effects of remifentanil and none obtained in non-mammalian animal models. The antinociceptive effects of remifentanil were assessed by using the acetic acid test in amphibians. Systemic and spinal administration of remifentanil was made by subcutaneous and intraspinal injections in the Northern grass frog, Rana pipiens. After administration, remifentanil produced dose-dependent and long-lasting antinociceptive effects which persisted for five hours after systemic administration but gave a shorter duration of action after spinal delivery. The antinociceptive effects of remifentanil were significantly blocked by pretreatment with systemic naltrexone. Systemic and spinal administration of remifentanil produced log dose-response curves which yielded ED50 values of 7.1 nmol/g and 3.2 nmol/animal respectively. The relative antinociceptive potency of remifentanil compared to other opioids administered to amphibians is similar to that found in mammalian models.