Oxytocin and Migraine Headache.

This article reviews material presented at the 2016 Scottsdale Headache Symposium. This presentation provided scientific results and rationale for the use of intranasal oxytocin for the treatment of migraine headache. Results from preclinical experiments are reviewed, including in vitro experiments demonstrating that trigeminal ganglia neurons possess oxytocin receptors and are inhibited by oxytocin. Furthermore, most of these same neurons contain CGRP, the release of which is inhibited by oxytocin. Results are also presented which demonstrate that nasal oxytocin inhibits responses of trigeminal nucleus caudalis neurons to noxious stimulation using either noxious facial shock or nitroglycerin infusion. These studies led to testing the analgesic effect of intranasal oxytocin in episodic migraineurs-studies which did not meet their primary endpoint of pain relief at 2 h, but which were highly informative and led to additional rat studies wherein inflammation was found to dramatically upregulate the number of oxytocin receptors available on trigeminal neurons. This importance of inflammation was supported by a series of in vivo rat behavioral studies, which demonstrated a clear craniofacial analgesic effect when a pre-existing inflammatory injury was present. The significance of inflammation was further solidified by a small single-dose clinical study, which showed analgesic efficacy that was substantially stronger in chronic migraine patients that had not taken an anti-inflammatory drug within 24 h of oxytocin dosing. A follow-on open label study examining effects of one month of intranasal oxytocin dosing did show a reduction in pain, but a more impressive decrease in the frequency of headaches in both chronic and high frequency episodic migraineurs. This study led to a multicountry double blind, placebo controlled study studying whether, over 2 months of dosing, "as needed" dosing of intranasal oxytocin by chronic and high frequency migraineurs would reduce the frequency of their headaches compared to a 1-month baseline period. This study failed to meet its primary endpoint, due to an extraordinarily high placebo rate in the country of most of the patients (Chile), but was also highly informative, showing strong results in other countries and strong post hoc indications of efficacy. The results provide a strong argument for further development of intranasal oxytocin for migraine prophylaxis.

Oxytocin receptor: Expression in the trigeminal nociceptive system and potential role in the treatment of headache disorders.

AIMS: Our studies investigated the location of oxytocin receptors in the peripheral trigeminal sensory system and determined their role in trigeminal pain. METHODS: Oxytocin receptor expression and co-localization with calcitonin gene-related peptide was investigated in rat trigeminal ganglion using immunohistochemistry. Enzyme-linked immunosorbent assay was used to determine the effects of facial electrocutaneous stimulation and adjuvant-induced inflammation of the temporomandibular joint on oxytocin receptor expression in the trigeminal ganglion. Finally, the effects of oxytocin on capsaicin-induced calcitonin gene-related peptide release from dural nociceptors were investigated using isolated rat dura mater. RESULTS: Oxytocin receptor immunoreactivity was present in rat trigeminal neurons. The vast majority of oxytocin receptor immunoreactive neurons co-expressed calcitonin gene-related peptide. Both electrocutaneous stimulation and adjuvant-induced inflammation led to a rapid upregulation of oxytocin receptor protein expression in trigeminal ganglion neurons. Oxytocin significantly and dose-dependently decreased capsaicin-induced calcitonin gene-related peptide release from dural nociceptors. CONCLUSION: Oxytocin receptor expression in calcitonin gene-related peptide containing trigeminal ganglion neurons, and the blockade of calcitonin gene-related peptide release from trigeminal dural afferents suggests that activation of these receptors may provide therapeutic benefit in patients with migraine and other primary headache disorders.

Positive Allosteric Modulation of A2AR Alters Immune Cell Responses and Ameliorates Psoriasis-Like Dermatitis in Mice.

Psoriasis is an immune cell‒mediated inflammatory disease of the skin with a mixed T helper type 1/T helper type17 cytokine environment combined with an innate immune response engaging toll-like receptors. Inflammatory diseases are characterized by dysregulated immune cell responses and elevated levels of adenosine at disease sites. Adenosine, acting through the A2AR, regulates inflammation, immune response, T-cell homeostasis, and tissue repair. We have identified a unique means to enhance A2AR function using a positive allosteric modulator. We show that oral administration of the A2AR-positive allosteric modulator AEA061 reduced ear swelling, skin thickness, erythema, scale formation, and inflammatory cytokine expression in A2Ar+/+ but not in A2Ar-/- mice with imiquimod-induced psoriasis-like dermatitis. Similar clinical and mRNA improvements were observed with topical administration. AEA061 also reduced clinical scores and cytokine expression in a mouse model of IL-23‒induced psoriasis-like dermatitis. In addition, AEA061 attenuated imiquimod-induced expression of IFN-α in plasmacytoid dendritic cells in vivo and IL-23 and IL-36α in conventional dendritic cells. TCR-mediated IL-17 expression in γδT cells in vivo and IL-17 production by CD4+ T cells enriched for γδT cells in vitro were also inhibited. Thus, the enhancement of A2AR responsiveness to the endogenous agonist adenosine through positive allosteric modulation is sufficient to enhance intrinsic homeostatic mechanisms attenuating disease progression.

EGT1442, a potent and selective SGLT2 inhibitor, attenuates blood glucose and HbA(1c) levels in db/db mice and prolongs the survival of stroke-prone rats.

Sodium glucose co-transporter 2 (SGLT2) is a renal type III integral membrane protein that co-transports sodium and glucose from filtrate to epithelium in the proximal tubule. Human subjects with homozygous or compound heterozygous mutations in SLC5A2 exhibit glucosuria without hypoglycemia or other obvious morbidity, suggesting that blockade of SGLT2 has the potential to promote normalization of blood glucose without hypoglycemia in the setting of type 2 diabetes. This report presents the in vitro and in vivo pharmacological activities of EGT1442, a recently discovered SGLT2 inhibitor in the C-aryl glucoside class. The inhibitory effects of EGT1442 for human SGLT1 and SGLT2 were evaluated in an AMG uptake assay and the in vivo efficacy of treatment with EGT1442 was investigated in rats and dogs after a single dose and in db/db mice after chronic administration. The effect of EGT1442 on median survival of SHRSP rats was also evaluated. The IC(50) values for EGT1442 against human SGLT1 and SGLT2 are 5.6µM and 2nM, respectively. In normal rats and dogs a saturable urinary glucose excretion was produced with an ED(50) of 0.38 and 0.09mg/kg, respectively. Following chronic administration to db/db mice, EGT1442 dose-dependently reduced HbA(1c) and blood glucose concentration without affecting body mass or insulin level. Additionally, EGT1442 significantly prolonged the median survival of SHRSP rats. EGT1442 showed favorable properties both in vitro and in vivo and could be beneficial to the management of type 2 diabetic patients.

Automated and Continuous Monitoring of Animal Welfare through Digital Alerting.

A primary goal in preclinical animal research is respectful and responsible care aimed toward minimizing stress and discomfort while enhancing collection of accurate and reproducible scientific data. Researchers use hands-on clinical observations and measurements as part of routine husbandry procedures or study protocols to monitor animal welfare. Although frequent assessments ensure the timely identification of animals with declining health, increased handling can result in additional stress on the animal and increased study variability. We investigated whether automated alerting regarding changes in behavior and physiology can complement existing welfare assessments to improve the identification of animals in pain or distress. Using historical data collected from a diverse range of therapeutic models, we developed algorithms that detect changes in motion and breathing rate frequently associated with sick animals but rare in healthy controls. To avoid introducing selec- tion bias, we evaluated the performance of these algorithms by using retrospective analysis of all studies occurring over a 31-d period in our vivarium. Analyses revealed that the majority of the automated alerts occurred prior to or simultaneously with technicians' observations of declining health in animals. Additional analyses performed across the entire duration of 2 studies (animal models of rapid aging and lung metastasis) demonstrated the sensitivity, accuracy, and utility of automated alerting for detecting unhealthy subjects and those eligible for humane endpoints. The percentage of alerts per total subject days ranged between 0% and 24%, depending on the animal model. Automated alerting effectively complements standard clinical observations to enhance animal welfare and promote responsible scientific advancement.

Retrospective Analysis of the Effects of Identification Procedures and Cage Changing by Using Data from Automated, Continuous Monitoring.

Many variables can influence animal behavior and physiology, potentially affecting scientific study outcomes. Laboratory and husbandry procedures-including handling, cage cleaning, injections, blood collection, and animal identification-may produce a multitude of effects. Previous studies have examined the effects of such procedures by making behavioral and physiologic measurements at specific time points; this approach can be disruptive and limits the frequency or duration of observations. Because these procedures can have both acute and long-term effects, the behavior and physiology of animals should be monitored continuously. We performed a retrospective data analysis on the effects of 2 routine procedures, animal identification and cage changing, on motion and breathing rates of mice continuously monitored in the home cage. Animal identification, specifically tail tattooing and ear tagging, as well as cage changing, produced distinct and reproducible postprocedural changes in spontaneous motion and breathing rate patterns. Behavioral and physiologic changes lasted approximately 2 d after tattooing or ear tagging and 2 to 4 d for cage changing. Furthermore, cage changes showed strain-, sex-, and time-of-day-dependent responses but not age-dependent differences. Finally, by reviewing data from a rodent model of multiple sclerosis as a retrospective case study, we documented that cage changing inadvertently affected experimental outcomes. In summary, we demonstrate how retrospective analysis of data collected continuously can provide high-throughput, meaningful, and longitudinal insights in to how animals respond to routine procedures.

Circulating microRNA and automated motion analysis as novel methods of assessing chemotherapy-induced peripheral neuropathy in mice.

Chemotherapy-induced peripheral neuropathy (CiPN) is a serious adverse effect in the clinic, but nonclinical assessment methods in animal studies are limited to labor intensive behavioral tests or semi-quantitative microscopic evaluation. Hence, microRNA (miRNA) biomarkers and automated in-life behavioral tracking were assessed for their utility as non-invasive methods. To address the lack of diagnostic biomarkers, we explored miR-124, miR-183 and miR-338 in a CiPN model induced by paclitaxel, a well-known neurotoxic agent. In addition, conventional and Vium's innovative Digital Vivarium technology-based in-life behavioral tests and postmortem microscopic examination of the dorsal root ganglion (DRG) and the sciatic nerve were performed. Terminal blood was collected on days 8 or 16, after 20 mg/kg paclitaxel was administered every other day for total of 4 or 7 doses, respectively, for plasma miRNA quantification by RT-qPCR. DRG and sciatic nerve samples were collected from mice sacrificed on day 16 for miRNA quantification. Among the three miRNAs analyzed, only miR-124 was statistically significantly increased (5 fold and 10 fold on day 8 and day 16, respectively). The increase in circulating miR-124 correlated with cold allodynia and axonal degeneration in both DRG and sciatic nerve. Automated home cage motion analysis revealed for the first time that nighttime motion was significantly decreased (P < 0.05) in paclitaxel-dosed animals. Although both increase in circulating miR-124 and decrease in nighttime motion are compelling, our results provide positive evidence warranting further testing using additional peripheral nerve toxicants and diverse experimental CiPN models.

Treatment with the atypical antipsychotic, olanzapine, prevents the expression of amphetamine-induced place conditioning in the rat.

Place conditioning (PC) experiments were conducted as a means to further elaborate the treatment potential of the atypical antipsychotic, olanzapine (OLZ), for stimulant abuse. The resulting preference/aversion provides an indirect measure of the incentive salience (i.e., euphoria/dysphoria) produced by a drug. Male Sprague-Dawley rats (n=48) were conditioned in two unique environments (i.e., vertical vs. horizontal stripped walls, large vs. small grid flooring) using injections (1.0 mg/kg ip) of either amphetamine (AMPH) or saline (SAL). On average, animals displayed a significant preference for the AMPH-paired location after 2.5 weeks of conditioning (five pairings each of AMPH and SAL). Once the preference was established, animals were pretreated (60 min) with a single dose of OLZ (0.0, 0.56, 1.0 or 1.5 mg/kg sc) given on the test (AMPH-free) day. For the following week's test, animals were injected with SAL (1.0 mg/kg ip) in an attempt to recapture the side preference exhibited before OLZ treatment. OLZ treatment prevented the expression of the AMPH-conditioned preference and reduced locomotor activity. Inhibition of preference resulted from the highest dose of OLZ (1.5 mg/kg), while the inhibition of locomotor activity occurred across all three doses. Additionally, while the effects on preference were no longer apparent by the SAL test the following week (reversible), the activity was still depressed during the SAL tests in animals that had experienced the highest dose of OLZ (1.5 mg/kg). Control experiments, in which OLZ was used as the conditioning drug, suggest that OLZ itself possesses no aversive effects in the PC paradigm, and may even produce a preference for the drug-paired chamber. Because the AMPH preference is dependent on dopamine (DA) release in the nucleus accumbens (NAcc), these experiments suggest that OLZ pretreatment interferes with the rewarding, as well as the subjective effects of AMPH.

Attenuation of the amphetamine discriminative cue in rats with the atypical antipsychotic olanzapine.

Sixteen male Sprague-Dawley rats were trained to discriminate between saline and amphetamine injections (1.0 mg/kg ip) using a standard two-lever (FR10) drug discrimination paradigm. A baseline dose-effect curve was generated for amphetamine administration alone, using doses both above and below the training dose (0.0-2.2 mg/kg ip). Once completed, a single dose of olanzapine (OLZ; 1.5 mg/kg sc) was tested for its ability to attenuate the amphetamine cue. OLZ pretreatment (60 min) successfully interfered with an animal's ability to discriminate amphetamine injections across various doses. The percentage of correct responding on the amphetamine lever and rate of responding were both significantly decreased across some but not all of the amphetamine doses. Therefore, we believe that this preliminary investigation has successfully shown that an OLZ dose of 1.5 mg/kg sc at 60 min can interfere with an animal's ability to detect some subjective cue(s) associated with amphetamine administration.

Involvement of the neurotensin receptor 1 in the behavioral effects of two neurotensin agonists, NT-2 and NT69L: lack of hypothermic, antinociceptive and antipsychotic actions in receptor knockout mice.

Neurotensin (NT) is a neuropeptide implicated in the pathophysiology of schizophrenia and in mediating the efficacy of antipsychotic drugs. NT is also involved in the regulation of body temperature and pain sensitivity. Using neurotensin receptor 1 (NTR1) knockout (KO) and wild-type (WT) mice, these studies evaluated the involvement of NTR1 in the behavioral responses produced by peripheral administration of NT agonists (NT-2 and NT69L). Animals were characterized in paradigms designed to assess hypothermia, antinociception, and antipsychotic-like effects. Under basal conditions, there were no phenotypic differences between NTR1 KO and WT mice. In WT mice, both NTR1 agonists decreased core body temperature (active doses in mg/kg, i.p., for NT-2 and NT69L, respectively: 1 and 3), increased tail withdrawal latencies (1 and 3), produced decreased spontaneous climbing (0.1, 0.3, 1 and 1, 3, 10) and reversed apomorphine-induced climbing (0.3, 1 and 1, 3). In contrast, none of the effects of either agonist were present in KO mice. These results suggest that NTR1: (1) does not play a major role in the control of basal thermoregulation, nociception or psychomotor stimulation in mice (barring possible developmental plasticity), (2) does mediate these behavioral responses to NT agonists, and (3) may play a role in the potential antipsychotic effects of these agonists.