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Background Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide pivotal in migraine pathophysiology and is considered a promising new migraine drug target. Although intravenous PACAP triggers migraine attacks and a recent phase II trial with a PACAP-inhibiting antibody showed efficacy in migraine prevention, targeting the PACAP receptor PAC1 alone has been unsuccessful. The present study investigated the role of three PACAP receptors (PAC1, VPAC1 and VPAC2) in inducing migraine-relevant hypersensitivity in mice. Methods Hindpaw hypersensitivity was induced by repeated PACAP38 injections. Tactile sensitivity responses were quantified using von Frey filaments in three knockout (KO) mouse strains, each lacking one of the PACAP-receptors (Ntotal = 160). Additionally, ex vivo wire myography was used to assess vasoactivity of the carotid artery, and gene expression of PACAP receptors was examined by qPCR. Results PACAP38 induced hypersensitivity in WT controls (p < 0.01) that was diminished in VPAC1 and VPAC2 KO mice (p < 0.05). In contrast, PAC1 KO mice showed similar responses to WT controls (p > 0.05). Myograph experiments supported these findings showing diminished vasoactivity in VPAC1 and VPAC2 KO mice. We found no upregulation of the non-modified PACAP receptors in KO mice. Conclusions This study assessed all three PACAP receptors in a migraine mouse model and suggests a significant role of VPAC receptors in migraine pathophysiology. The lack of hypersensitivity reduction in PAC1 KO mice suggests the involvement of other PACAP receptors or compensatory mechanisms. The results indicate that targeting only individual PACAP receptors may not be an effective migraine treatment., BACKGROUND: Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide pivotal in migraine pathophysiology and is considered a promising new migraine drug target. Although intravenous PACAP triggers migraine attacks and a recent phase II trial with a PACAP-inhibiting antibody showed efficacy in migraine prevention, targeting the PACAP receptor PAC1 alone has been unsuccessful. The present study investigated the role of three PACAP receptors (PAC1, VPAC1 and VPAC2) in inducing migraine-relevant hypersensitivity in mice. METHODS: Hindpaw hypersensitivity was induced by repeated PACAP38 injections. Tactile sensitivity responses were quantified using von Frey filaments in three knockout (KO) mouse strains, each lacking one of the PACAP-receptors (Ntotal = 160). Additionally, ex vivo wire myography was used to assess vasoactivity of the carotid artery, and gene expression of PACAP receptors was examined by qPCR. RESULTS: PACAP38 induced hypersensitivity in WT controls (p < 0.01) that was diminished in VPAC1 and VPAC2 KO mice (p < 0.05). In contrast, PAC1 KO mice showed similar responses to WT controls (p > 0.05). Myograph experiments supported these findings showing diminished vasoactivity in VPAC1 and VPAC2 KO mice. We found no upregulation of the non-modified PACAP receptors in KO mice. CONCLUSIONS: This study assessed all three PACAP receptors in a migraine mouse model and suggests a significant role of VPAC receptors in migraine pathophysiology. The lack of hypersensitivity reduction in PAC1 KO mice suggests the involvement of other PACAP receptors or compensatory mechanisms. The results indicate that targeting only individual PACAP receptors may not be an effective migraine treatment.

Ibuprofen (IBU) er blandt de mest anvendte milde analgetika i Europa og USA og er første valg når der anbefales non-steroide anti-inflammatoriske lægemidler (NSAID). IBU bliver benyttet af mange gravide kvinder gennem hele graviditeten, selvom brugen af IBU er kontraindiceret i tredje trimester. Dette er bekymrende, fordi IBU har vist antiandrogene effekter og flere studier har påvist sammenhænge mellem brugen af IBU og hormonforstyrrende effekter. Brugen af IBU under graviditet har vist sig at føre til øget risiko for reproduktive forstyrrelser, såsom Testikulært Dysgenese Syndrom (TDS) i mænd og Ovarie Dysgenese Syndrom (ODS) i kvinder. Litteraturen foreslår at IBU’s antiandrogene effekt skyldes at CYP17-enzymet inhiberes. Formålet med dette speciale var at undersøge effekterne af intrauterin IBU-eksponering på steroidgenesen i reproduktive organer (testikler, ovarier, uterin horn og fundus) samt binyrer og hypofyser i B6 mus. Dette blev undersøgt ved brug af en dyremodel, hvor gravide mus blev eksponeret for IBU (150 mg/kg/dag) under hele graviditeten. Steroid niveauer i væv fra afkommet blev kvantificeret ved brug af LC-MS/MS. Derudover blev den steroidogene genekspression i gonaderne undersøgt ved brug af RT-qPCR og luteiniserende hormon (LH) koncentrationer i plasma prøver bestemt ved ELISA. Resultaterne fra dette studie viste at IBU-eksponering under graviditeten resulterede i et signifikant fald i antal unger fra 5,6 unger til 3,3 unger. Den anogenitale distance (AGD) blev målt på alle unger i postnatal uge 12 og blev brugt som biomarkør til at bestemme mangel på androgener under fosterudviklingen. Dog blev der ikke observeret nogle signifikante effekter på AGD for hverken hanner eller hunner, hvilket stemmer overens med steroidprofilerne fra hannerne, hvor der blev observeret en betydelig stigning i androgener, især testosteron (TS) og dihydrotestosteron (DHT). En hypotese om hvorvidt stigningen i TS-niveauer kunne være et resultat fra IBU-induceret, Ibuprofen (IBU) is one of the most frequently used mild analgesics in Europe and USA and is first-line treatment when non-steroidal anti-inflammatory drugs (NSAID) are recommended. Even though use of IBU in third trimester is contraindicated, IBU is still consumed by many pregnant women throughout pregnancy. This is of concern as IBU has shown antiandrogenic effects, and several studies have demonstrated associations between IBU consumption and endocrine disrupting effects. The use of IBU during pregnancy have shown to increase the risk of reproductive disorders, such as Testicular Dysgenesis Syndrome (TDS) in men and a corresponding condition in women, known as Ovarian Dysgenesis Syndrome (ODS). The literature suggest that CYP17-inhibition may be responsible for IBU’s antiandrogenic effect. The aim of this master thesis was to determine the effects of intrauterine IBU exposure on steroidogenesis in reproductive organs (testes, ovaries, uterine horn, fundus), as well as adrenal and pituitary glands in B6 mice. This was investigated using an animal model, where pregnant mice were exposed to IBU (150 mg/kg/day) throughout the entire pregnancy. The steroid levels in tissues from offspring were then quantified using LC-MS/MS. Additionally, steroidogenic gene expression in gonads was investigated using RT-qPCR, and luteinizing hormone (LH) levels in plasma samples were measured using ELISA. Findings from this study demonstrated that IBU exposure during pregnancy resulted in a significantly reduced litter size from 5.6 pups to 3.3 pups. The anogenital distance (AGD) was measured on all offspring at postnatal week 12 and used as a biomarker to determine deficiency of androgens during fetal development. However, no significant effects were observed in AGD in either males nor females, which corresponds with the steroid profile in males, where a significant increase in androgens, especially testosterone (TS) and dihydrotestosterone (DHT) was observed. The increase in

Cytoglobin is a heme protein with unresolved physiological function. Genetic deletion of zebrafish cytoglobin (cygb2) causes developmental defects in left-right cardiac determination, which in humans is associated with defects in ciliary function and low airway epithelial nitric oxide production. Here we show that Cygb2 co-localizes with cilia and with the nitric oxide synthase Nos2b in the zebrafish Kupffer’s vesicle, and that cilia structure and function are disrupted in cygb2 mutants. Abnormal ciliary function and organ laterality defects are phenocopied by depletion of nos2b and of gucy1a, the soluble guanylate cyclase homolog in fish. The defects are rescued by exposing cygb2 mutant embryos to a nitric oxide donor or a soluble guanylate cyclase stimulator, or with over-expression of nos2b. Cytoglobin knockout mice also show impaired airway epithelial cilia structure and reduced nitric oxide levels. Altogether, our data suggest that cytoglobin is a positive regulator of a signaling axis composed of nitric oxide synthase–soluble guanylate cyclase–cyclic GMP that is necessary for normal cilia motility and left-right patterning.

Purpose: In this study, we used broadband near-infrared spectroscopy, a non-invasive optical technique, to investigate in real time the possible role of neuroglobin in retinal hemodynamics and metabolism. Methods: Retinae of 12 C57 mice (seven young and five old) and seven young neuroglobin knockouts (Ngb-KOs) were exposed to light from a low-power halogen source, and the back-reflected light was used to calculate changes in the concentration of oxygenated hemoglobin (HbO2), deoxygenated hemoglobin (HHb), and oxidized cytochrome c oxidase (oxCCO). Results: The degree of change in the near-infrared spectroscopy signals associated with HHb, HbO2, and oxCCO was significantly greater in young C57 mice compared to the old C57 mice (P < 0.05) and the Ngb-KO model (P < 0.005). Conclusions: Our results reveal a possible role of Ngb in regulating retinal function, as its absence in the retinae of a knockout mouse model led to suppressed signals that are associated with hemodynamics and oxidative metabolism. Translational Relevance: Near-infrared spectroscopy enabled the non-invasive detection of characteristic signals that differentiate between the retina of a neuroglobin knockout mouse model and that of a wild-type model. Further work is needed to evaluate the source of the signal differences and how these differences relate to the presence or absence of neuroglobin in the ganglion, bipolar, or amacrine cells of the retina.

Background Calcitonin gene-related peptide (CGRP) antagonizing drugs represents the most important advance in migraine therapy for decades. However, these new drugs are only effective in 50-60% of patients. Recent studies have shown that the pituitary adenylate cyclase-activating peptide (PACAP38) pathway is independent from the CGRP signaling pathway. Here, we investigate PACAP38 signaling pathways in relation to glyceryl trinitrate (GTN), levcromakalim and sumatriptan. Methods In vivo mouse models of PACAP38-, GTN-, and levcromakalim-induced migraine were applied using tactile sensitivity to von Frey filaments as measuring readout. Signaling pathways involved in the three models were dissected using PACAP-inhibiting antibodies (mAbs) and sumatriptan. Results We showed that PACAP mAbs block PACAP38 induced hypersensitivity, but not via signaling pathways involved in GTN and levcromakalim. Also, sumatriptan has no effect on PACAP38-induced hypersensitivity relevant to migraine. This is the first study testing the effect of a PACAP-inhibiting drug on GTN- and levcromakalim-induced hypersensitivity. Conclusions Based on the findings in our mouse model of migraine using migraine-inducing compounds and anti-migraine drugs, we suggest that PACAP acts via a distinct pathway. Using PACAP38 antagonism may be a novel therapeutic target of interest in a subgroup of migraine patients who do not respond to existing therapies.

Neuroglobin (Ngb) is found in the neurones of several different brain areas and is known to bind oxygen and other gaseous molecules and reactive oxygen species (ROS) in vitro, but it does not seem to act as a respiratory molecule for neurones. Using male and female Ngb-knockout (KO) mice, we addressed the role of Ngb in neuronal brain activity using behavioral tests but found no differences in general behaviors, memory processes, and anxiety-/depression-like behaviors. Oxidative stress and ROS play key roles in epileptogenesis, and oxidative injury produced by an excessive production of free radicals is involved in the initiation and progression of epilepsy. The ROS binding properties led us to hypothesize that lack of Ngb could affect central coping with excitatory stimuli. We consequently explored whether exposure to the excitatory molecule kainate (KA) would increase severity of seizures in mice lacking Ngb. We found that the duration and severity of seizures were increased, while the latency time to develop seizures was shortened in Ngb-KO compared to wildtype adult female mice. Consistently, c-fos expression after KA was significantly increased in Ngb-KO mice in the amygdala and piriform cortex, regions rich in Ngb and known to be centrally involved in seizure generation. Moreover, the measured c-fos expression levels were correlated with seizure susceptibility. With these new findings combined with previous studies we propose that Ngb could constitute an intrinsic defense mechanism against neuronal hyperexcitability and oxidative stress by buffering of ROS in amygdala and other Ngb-containing brain regions.