Wound Healing in a Porcine Model of Retinal Holes.

Purpose: To investigate retinal wound healing, we created a new porcine model of retinal hole and identified the cells involved in hole closure. Methods: Sixteen landrace pigs underwent vitrectomy, and a subretinal bleb was created before cutting a retinal hole using a 23G vitrector. No tamponade was used. Before surgery and one, two, and four weeks after surgery, the eyes were examined by optical coherence tomography and color fundus photos. At the end of follow-up, the eyes were enucleated for histology. Tissue sections of 5 µm were prepared for hematoxylin-eosin staining and immunohistochemical analysis with antibodies to retinal glial and epithelial cells. Results: Retinal holes below 1380 µm in diameter closed spontaneously within four weeks, whereas larger holes remained open. Hole closure was mediated by central movement of the edges of the hole and in most cases the formation of a gliotic plug. Fluorescence microscopy revealed that the plug consisted of cells positive for glial fibrillary acidic protein, indicating the presence of macroglial cell types. Specifically, the plug was positive for S100 calcium-binding protein B, mainly representing astrocytes, while it was negative for anti-glutamine syntethase, representing Müller glia. These findings suggest that astrocytes are the predominating cell type in the plug. Minimal glial reaction was seen in the retinal holes that did not close. Conclusions: We present a new porcine model for investigating large retinal holes. The retinal holes closed by approximation of hole edges, and the remnant retinal defect was closed with an astroglial plug.

VPAC1 and VPAC2 receptors mediate tactile hindpaw hypersensitivity and carotid artery dilatation induced by PACAP38 in a migraine relevant mouse model.

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.

Successful liver transplantation as rescue therapy in a patient with metastases from a vasoactive intestinal peptide producing neuroendocrine tumor.

This case report presents a 40-year-old patient with a vasoactive intestinal peptide (VIP) secreting high grade (Ki-67 39%) neuroendocrine tumor (NET) from the pancreas, for whom successful liver transplantation (LT) was carried out 8 years after resection of the primary tumor due to massive liver metastases. The transplantation was done as rescue therapy due to rapid progression and a devastating clinical condition requiring intravenous supplementation for 20 hours daily. The latest imaging carried out 18 months after transplantation is without signs of recurrence, and the patient is in good health with undetectable levels of VIP. According to the guidelines, LT is only recommended if Ki-67 is <20% and if there has been tumor control for more than 6 months prior to transplantation. Our case illustrates that LT is an option that should be considered for selected NET patients without extrahepatic involvement regardless of tumor grade and clinical condition.

Glucagon does not directly stimulate pituitary secretion of ACTH, GH or copeptin.

Glucagon is best known for its contribution to glucose regulation through activation of the glucagon receptor (GCGR), primarily located in the liver. However, glucagon's impact on other organs may also contribute to its potent effects in health and disease. Given that glucagon-based medicine is entering the arena of anti-obesity drugs, elucidating extrahepatic actions of glucagon are of increased importance. It has been reported that glucagon may stimulate secretion of arginine-vasopressin (AVP)/copeptin, growth hormone (GH) and adrenocorticotrophic hormone (ACTH) from the pituitary gland. Nevertheless, the mechanisms and whether GCGR is present in human pituitary are unknown. In this study we found that intravenous administration of 0.2 mg glucagon to 14 healthy subjects was not associated with increases in plasma concentrations of copeptin, GH, ACTH or cortisol over a 120-min period. GCGR immunoreactivity was present in the anterior pituitary but not in cells containing GH or ACTH. Collectively, glucagon may not directly stimulate secretion of GH, ACTH or AVP/copeptin in humans but may instead be involved in yet unidentified pituitary functions.

Secretion of glucagon, GLP-1 and GIP may be affected by circadian rhythm in healthy males.

BACKGROUND: Glucagon is secreted from pancreatic alpha cells in response to low blood glucose and increases hepatic glucose production. Furthermore, glucagon enhances hepatic protein and lipid metabolism during a mixed meal. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are secreted from gut endocrine cells during meals and control glucose homeostasis by potentiating insulin secretion and inhibiting food intake. Both glucose homeostasis and food intake have been reported to be affected by circadian rhythms and vice versa. In this study, we investigated whether the secretion of glucagon, GLP-1 and GIP was affected by circadian rhythms. METHODS: A total of 24 healthy men with regular sleep schedules were examined for 24 h at the hospital ward with 15 h of wakefulness and 9 h of sleep. Food intake was standardized, and blood samples were obtained every third hour. Plasma concentrations of glucagon, GLP-1 and GIP were measured, and data were analyzed by rhythmometric statistical methods. Available data on plasma glucose and plasma C-peptide were also included. RESULTS: Plasma concentrations of glucagon, GLP-1, GIP, C-peptide and glucose fluctuated with a diurnal 24-h rhythm, with the highest levels during the day and the lowest levels during the night: glucagon (p < 0.0001, peak time 18:26 h), GLP-1 (p < 0.0001, peak time 17:28 h), GIP (p < 0.0001, peak time 18:01 h), C-peptide (p < 0.0001, peak time 17.59 h), and glucose (p < 0.0001, peak time 23:26 h). As expected, we found significant correlations between plasma concentrations of C-peptide and GLP-1 and GIP but did not find correlations between glucose concentrations and concentrations of glucagon, GLP-1 and GIP. CONCLUSIONS: Our results demonstrate that under meal conditions that are similar to that of many free-living individuals, plasma concentrations of glucagon, GLP-1 and GIP were observed to be higher during daytime and evening than overnight. These findings underpin disturbed circadian rhythm as a potential risk factor for diabetes and obesity. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT06166368. Registered 12 December 2023.

Development of a New Enzyme-Linked Immunosorbent Assay (ELISA) for Measuring the Content of PACAP in Mammalian Tissue and Plasma.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a naturally occurring neuropeptide found in both the central and peripheral nervous systems of vertebrates. Recent studies have revealed the presence of PACAP and its corresponding receptors, namely, the pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R), vasoactive intestinal peptide receptor 1 (VIPR1), and vasoactive intestinal peptide receptor 2 (VIPR2), in various structures implicated in migraine pathophysiology, including sensory trigeminal neurons. Human studies have demonstrated that when infused, PACAP can cause dilation of cranial vessels and result in delayed migraine-like attacks. In light of this, we present a novel ELISA assay that has been validated for quantifying PACAP in tissue extracts and human plasma. Using two well characterized antibodies specifically targeting PACAP, we successfully developed a sandwich ELISA assay, capable of detecting and accurately quantifying PACAP without any cross-reactivity to closely related peptides. The quantification range was between 5.2 pmol/L and 400 pmol/L. The recovery in plasma ranged from 98.2% to 100%. The increasing evidence pointing to the crucial role of PACAP in migraine pathophysiology necessitates the availability of tools capable of detecting changes in the circulatory levels of PACAP and its potential application as a reliable biomarker.

Plasma levels of VIP are not elevated during PACAP- and VIP-induced cluster headache attacks: an exploratory study.

Background: Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) provoked cluster headache attacks in individuals with episodic cluster headache during their active phase and individuals with chronic cluster headache. In this study, we investigated whether infusions of PACAP and VIP caused alterations in plasma levels of VIP and their potential contribution to induced cluster headache attacks. Methods: Participants received either PACAP or VIP infusion for 20 min on 2 separate days with an interval of at least 7 days in between. Blood collection was performed at T0, T20, T30, and T90. Plasma levels of VIP were measured using a validated radioimmunoassay method. Results: Blood samples were collected from participants with episodic cluster headache in the active phase (eCHA, n = 14), remission (eCHR, n = 15), and from participants with chronic cluster headache (cCH, n = 15). Baseline levels of VIP did not differ among the three groups (p = 0.1161). During PACAP infusion, mixed-effects analysis revealed a significant increase in plasma levels of VIP in eCHA (p = 0.0300) and eCHR (p = 0.0058) but not in cCH (p = 0.2930). We found no difference in the increase of plasma VIP levels between patients who developed PACAP38- or VIP-induced attacks. Conclusion: Cluster headache attacks induced by PACAP38 or VIP infusion are not associated with changes in plasma levels of VIP. Further studies are needed to investigate the role of VIP and the parasympathetic system in cluster headache. Clinical trial registration: The parent study is registered at ClinicalTrials.gov (NCT03814226).

Larger interface area at the human myotendinous junction in type 1 compared with type 2 muscle fibers.

The myotendinous junction (MTJ) is structurally specialized to transmit force. The highly folded muscle membrane at the MTJ increases the contact area between muscle and tendon and potentially the load tolerance of the MTJ. Muscles with a high content of type II fibers are more often subject to strain injury compared with muscles with type I fibers. It is hypothesized that this is explained by a smaller interface area of MTJ in type II compared with type I muscle fibers. The aim was to investigate by confocal microscopy whether there is difference in the surface area at the MTJ between type I and II muscle fibers. Individual muscle fibers with an intact MTJ were isolated by microscopic dissection in samples from human semitendinosus, and they were labeled with antibodies against collagen XXII (indicating MTJ) and type I myosin (MHCI). Using a spinning disc confocal microscope, the MTJ from each fiber was scanned and subsequently reconstructed to a 3D-model. The interface area between muscle and tendon was calculated in type I and II fibers from these reconstructions. The MTJ was analyzed in 314 muscle fibers. Type I muscle fibers had a 22% larger MTJ interface area compared with type II fibers (p < 0.05), also when the area was normalized to fiber diameter. By the new method, it was possible to analyze the structure of the MTJ from a large number of human muscle fibers. The finding that the interface area between muscle and tendon is higher in type I compared with type II fibers suggests that type II fibers are less resistant to strain and therefore more susceptible to injury.

Phenotyping of light-activated neurons in the mouse SCN based on the expression of FOS and EGR1.

Light-sensitive neurons are located in the ventral and central core of the suprachiasmatic nucleus (SCN), whereas stably oscillating clock neurons are found mainly in the dorsal shell. Signals between the SCN core and shell are believed to play an important role in light entrainment. Core neurons express vasoactive intestinal polypeptide (VIP), gastrin-releasing peptide (GRP), and Neuroglobin (Ngb), whereas the shell neurons express vasopressin (AVP), prokineticin 2, and the VIP type 2 (VPAC2) receptor. In rodents, light has a phase-shifting capacity at night, which induces rapid and transient expression of the EGR1 and FOS in the SCN. Methods: The present study used immunohistochemical staining of FOS, EGR1, and phenotypical markers of SCN neurons (VIP, AVP, Ngb) to identify subtypes/populations of light-responsive neurons at early night. Results: Double immunohistochemistry and cell counting were used to evaluate the number of SCN neurons expressing FOS and EGR1 in the SCN. The number of neurons expressing either EGR1 or FOS was higher than the total number of neurons co-storing EGR1 and FOS. Of the total number of light-responsive cells, 42% expressed only EGR1, 43% expressed only FOS, and 15% expressed both EGR1 and FOS. Light-responsive VIP neurons represented only 31% of all VIP neurons, and EGR1 represents the largest group of light-responsive VIP neurons (18%). VIP neurons expressing only FOS represented 1% of the total light-responsive VIP neurons. 81% of the Ngb neurons in the mouse SCN were light-responsive, and of these neurons expressing only EGR1 after light stimulation represented 44%, whereas 24% expressed FOS. Although most light-responsive neurons are found in the core of the SCN, 29% of the AVP neurons in the shell were light-responsive, of which 8% expressed EGR1, 10% expressed FOS, and 11% co-expressed both EGR1 and FOS after light stimulation. Discussion: Our analysis revealed cell-specific differences in light responsiveness between different peptidergic and Ngb-expressing neurons in different compartments of the mouse SCN, indicating that light activates diverse neuronal networks in the SCN, some of which participate in photoentrainment.

Flavobacterium psychrophilum: Response of Vaccinated Large Rainbow Trout to Different Strains.

BACKGROUND: Although Flavobacterium psychrophilum is recognized as the causative pathogen of rainbow trout fry syndrome (RTFS), often resulting in high fry mortality, it is also responsible for bacterial cold water disease (BCWD) in large and older rainbow trout (Oncorhynchus mykiss). These older fish do not experience high mortality, but sustain, through the shedding of bacteria, a constant infection pressure at farm level, which exposes fry to an unnecessary infection risk. We have produced and assessed the immunogenicity of an experimental injection BCWD vaccine, which may be used to decrease the shedding of bacteria from older fish. METHODS: A total of 800 fish were i.p.-injected: 200 fish received the bacterin with adjuvant, 200 fish received the bacterin alone, 200 fish received adjuvant alone and 200 fish were injected with physiological saline. Blood samples were taken at day 0 and at three different time points (4, 8 and 14 weeks) post-vaccination. Plasma antibody levels were measured by ELISA for reactivity against both the homologous F. psychrophilum vaccine strain (serotype Fd) and heterologous strains (serotype Th). RESULTS: Significantly elevated antibody titers were found against all serotypes in vaccinated fish. Welfare parameters associated with the vaccination process were evaluated by analyzing trout plasma samples for six different biochemical parameters, but no adverse effects associated with injection were indicated. CONCLUSIONS: The study suggests that an injection vaccine containing formalin-inactivated whole cells of F. psychrophilum (serotype Fd), adjuvanted with FIA, may also induce protection against heterologous strains. We advocate for, as the next step, the performance of field trials evaluating if the vaccination of older rainbow trout will (1) reduce the infection pressure in farms, (2) elevate the general health level in all groups and (3) minimize F. psychrophilum infection in fry at farm level. This may reduce the need for the administration of antibiotics in all age classes.

Localization of the neuropeptides pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal peptide, and their receptors in the basal brain blood vessels and trigeminal ganglion of the mouse CNS; an immunohistochemical study.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are structurally related neuropeptides that are widely expressed in vertebrate tissues. The two neuropeptides are pleiotropic and have been associated with migraine pathology. Three PACAP and VIP receptors have been described: PAC1, VPAC1, and VPAC2. The localization of these receptors in relation to VIP and PACAP in migraine-relevant structures has not previously been shown in mice. In the present study, we used fluorescence immunohistochemistry, well-characterized antibodies, confocal microscopy, and three-dimensional reconstruction to visualize the distribution of PACAP, VIP, and their receptors in the basal blood vessels (circle of Willis), trigeminal ganglion, and brain stem spinal trigeminal nucleus (SP5) of the mouse CNS. We demonstrated a dense network of circularly oriented VIP fibers on the basal blood vessels. PACAP nerve fibers were fewer in numbers compared to VIP fibers and ran along the long axis of the blood vessels, colocalized with calcitonin gene-related peptide (CGRP). The nerve fibers expressing CGRP are believed to be sensorial, with neuronal somas localized in the trigeminal ganglion and PACAP was found in a subpopulation of these CGRP-neurons. Immunostaining of the receptors revealed that only the VPAC1 receptor was present in the basal blood vessels, localized on the surface cell membrane of vascular smooth muscle cells and innervated by VIP fibers. No staining was seen for the PAC1, VPAC1, or VPAC2 receptor in the trigeminal ganglion. However, distinct PAC1 immunoreactivity was found in neurons innervated by PACAP nerve terminals located in the spinal trigeminal nucleus. These findings indicate that the effect of VIP is mediated via the VPAC1 receptor in the basal arteries. The role of PACAP in cerebral arteries is less clear. The localization of PACAP in a subpopulation of CGRP-expressing neurons in the trigeminal ganglion points toward a primary sensory function although a dendritic release cannot be excluded which could stimulate the VPAC1 receptor or the PAC1 and VPAC2 receptors on immune cells in the meninges, initiating neurogenic inflammation relevant for migraine pathology.

Prevention of Cell Death by Activation of Hydroxycarboxylic Acid Receptor 1 (GPR81) in Retinal Explants.

BACKGROUND: Progressive retinal ganglion cell (RGC) dysfunction and death are common characteristics of retinal neurodegenerative diseases. Recently, hydroxycarboxylic acid receptor 1 (HCA1R, GPR81) was identified as a key modulator of mitochondrial function and cell survival. Thus, we aimed to test whether activation of HCA1R with 3,5-Dihydroxybenzoic acid (DHBA) also promotes RGC survival and improves energy metabolism in mouse retinas. METHODS: Retinal explants were treated with 5 mM of the HCA1R agonist, 3,5-DHBA, for 2, 4, 24, and 72 h. Additionally, explants were also treated with 15 mM of L-glutamate to induce toxicity. Tissue survival was assessed through lactate dehydrogenase (LDH) viability assays. RGC survival was measured through immunohistochemical (IHC) staining. Total ATP levels were quantified through bioluminescence assays. Energy metabolism was investigated through stable isotope labeling and gas chromatography-mass spectrometry (GC-MS). Lactate and nitric oxide levels were measured through colorimetric assays. RESULTS: HCA1R activation with 3,5-DHBAincreased retinal explant survival. During glutamate-induced death, 3,5-DHBA treatment also increased survival. IHC analysis revealed that 3,5-DHBA treatment promoted RGC survival in retinal wholemounts. 3,5-DHBA treatment also enhanced ATP levels in retinal explants, whereas lactate levels decreased. No effects on glucose metabolism were observed, but small changes in lactate metabolism were found. Nitric oxide levels remained unaltered in response to 3,5-DHBA treatment. CONCLUSION: The present study reveals that activation of HCA1R with 3,5-DHBA treatment has a neuroprotective effect specifically on RGCs and on glutamate-induced retinal degeneration. Hence, HCA1R agonist administration may be a potential new strategy for rescuing RGCs, ultimately preventing visual disability.

Plasma Levels of CGRP During a 2-h Infusion of VIP in Healthy Volunteers and Patients With Migraine: An Exploratory Study.

Introduction: The activation of perivascular fibers and the consequent release of vasoactive peptides, including the vasoactive intestinal polypeptide (VIP), play a role in migraine pathogenesis. A 2-h infusion of VIP provoked migraine, but the mechanisms remain unknown. We investigated whether 2-h infusion of VIP caused alterations in plasma levels of the calcitonin gene-related peptide (CGRP) and whether any changes might be related to the induced migraine attacks. Materials and Methods: We enrolled individuals with episodic migraine without aura and healthy participants to randomly receive a 2-h infusion of either VIP (8 pmol/kg/min) or placebo (sterile saline) in two randomized, placebo-controlled crossover trials. We collected clinical data and measured plasma levels of VIP and CGRP at fixed time points: at baseline (T0) and every 30 min until 180 min (T180) after the start of the infusion. Results: Blood samples were collected from patients with migraine (n = 19) and healthy individuals (n = 12). During VIP infusion, mixed effects analysis revealed a significant increase in plasma CGRP (p = 0.027) at T30 (vs. T180, adjusted p-value = 0.039) and T60 (vs. T180, adjusted p-value = 0.027) in patients with migraine. We found no increase in plasma CGRP during VIP-induced migraine attacks (p = 0.219). In healthy individuals, there was no increase in plasma CGRP during VIP (p = 0.205) or placebo (p = 0.428) days. Discussion: Plasma CGRP was elevated in patients with migraine during a prolonged infusion of VIP, but these alterations were not associated with VIP-induced migraine attacks. Given the exploratory design of our study, further investigations are needed to clarify the role of CGRP in VIP-induced migraine. Clinical Trial Registration: ClinicalTrials.gov, identifier: NCT03989817 and NCT04260035.

Circadian regulation of protein cargo in extracellular vesicles.

The circadian clock controls many aspects of physiology, but it remains undescribed whether extracellular vesicles (EVs), including exosomes, involved in cell-cell communications between tissues are regulated in a circadian pattern. We demonstrate a 24-hour rhythmic abundance of individual proteins in small EVs using liquid chromatography-mass spectrometry in circadian-synchronized tendon fibroblasts. Furthermore, the release of small EVs enriched in RNA binding proteins was temporally separated from those enriched in cytoskeletal and matrix proteins, which peaked during the end of the light phase. Last, we targeted the protein sorting mechanism in the exosome biogenesis pathway and established (by knockdown of circadian-regulated flotillin-1) that matrix metalloproteinase 14 abundance in tendon fibroblast small EVs is under flotillin-1 regulation. In conclusion, we have identified proteomic time signatures for small EVs released by tendon fibroblasts, which supports the view that the circadian clock regulates protein cargo in EVs involved in cell-cell cross-talk.

Decreased Glucose Metabolism and Glutamine Synthesis in the Retina of a Transgenic Mouse Model of Alzheimer's Disease.

Visual changes are some of the earliest symptoms that patients with Alzheimer's disease (AD) experience. Pathophysiological processes such as amyloid-ß plaque formation, vascular changes, neuroinflammation, and loss of retinal ganglion cells (RGCs) have been detected in the retina of AD patients and animal models. However, little is known about the molecular processes that underlie retinal neurodegeneration in AD. The cellular architecture and constant sensory activity of the retina impose high metabolic demands. We thus hypothesized that energy metabolism might be compromised in the AD retina similarly to what has been observed in the AD brain. To address this question, we explored cellular alterations and retinal metabolic activity in the 5 × FAD mouse model of AD. We used 8-month-old female 5 × FAD mice, in which the AD-related pathology has been shown to be apparent. We observed that RGC density is selectively affected in the retina of 5 × FAD mice. To map retinal metabolic activity, we incubated isolated retinal tissue with [U-13C] glucose and analyzed tissue extracts by gas chromatography-mass spectrometry. We found that the retinas of 5 × FAD mice exhibit glucose hypometabolism. Moreover, we detected decreased glutamine synthesis in 5 × FAD retinas but no changes in the expression of markers of Müller glia, the main glial cell type responsible for glutamate uptake and glutamine synthesis in the retina. These findings suggest that AD presents with metabolic alterations not only in the brain but also in the retina that may be detrimental to RGC activity and survival, potentially leading to the visual impairments that AD patients suffer.

The Circadian Clock Is Sustained in the Thyroid Gland of VIP Receptor 2 Deficient Mice.

VIP/VPAC2-receptor signaling is crucial for functioning of the circadian clock in the suprachiasmatic nucleus (SCN) since the lack results in disrupted synchrony between SCN cells and altered locomotor activity, body temperature, hormone secretion and heart rhythm. Endocrine glands, including the thyroid, show daily oscillations in clock gene expression and hormone secretion, and SCN projections target neurosecretory hypothalamic thyroid-stimulating hormone (TSH)-releasing hormone cells. The aim of the study was to gain knowledge of mechanisms important for regulation of the thyroid clock by evaluating the impact of VIP/VPAC2-receptor signaling. Quantifications of mRNAs of three clock genes (Per1, Per2 and Bmal1) in thyroids of wild type (WT) and VPAC2-receptor deficient mice were done by qPCR. Tissues were taken every 4th h during 24-h 12:12 light-dark (LD) and constant darkness (DD) periods, both genders were used. PER1 immunoreactivity was visualized on sections of both WT and VPAC2 lacking mice during a LD cycle. Finally, TSH and the thyroid hormone T4 levels were measured in the sera by commercial ELISAs. During LD, rhythmic expression of all three mRNA was found in both the WT and knockout animals. In VPAC2-receptor knockout animals, the amplitudes were approximately halved compared to the ones in the WT mice. In the WT, Per1 mRNA peaked around "sunset", Per2 mRNA followed with approximately 2 h, while Bmal1 mRNA was in antiphase with Per1. In the VPAC2 knockout mice, the phases of the mRNAs were advanced approximately 5 h compared to the WT. During DD, the phases of all the mRNAs were identical to the ones found during LD in both groups of mice. PER1 immunoreactivity was delayed compared to its mRNA and peaked during the night in follicular cells of both the thyroid and parathyroid glands in the WT animals. In WT animals, TSH was high around the transition to darkness compared to light-on, while T4 did not change during the 24 h cycle. In conclusion, sustained and identical rhythms (phases and amplitudes) of three clock genes were found in VPAC2 deficient mice during LD and DD suggesting high degree of independence of the thyroid clock from the master SCN clock.

Effect of Vasoactive Intestinal Polypeptide on Development of Migraine Headaches: A Randomized Clinical Trial.

Importance: Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptides (PACAPs) are structurally and functionally related, yet different in their migraine-inducing properties. It remains unclear whether the lack of migraine induction can be attributed to the only transient vasodilatory response after a 20-minute infusion of VIP. Objective: To determine whether a 2-hour infusion of VIP would provoke migraine attacks. Design, Setting, and Participants: A randomized, double-blind, placebo-controlled, crossover study was conducted between May and September 2020 at the Danish Headache Center in Copenhagen, Denmark. Patients were eligible for inclusion if they were ages 18 to 40 years, weighed between 50 and 90 kg, had a diagnosis of migraine without aura as defined by the International Classification of Headache Disorders, and had a migraine frequency of 1 to 6 attacks per month. Interventions: Patients were randomly allocated to receive a 2-hour infusion of VIP or placebo on 2 different days. Main Outcomes and Measures: The primary end point was the difference in incidence of experimentally induced migraine attacks during the observational period (0-12 hours) between VIP and placebo. Results: Twenty-one patients (17 [81%] women and 4 [19%] men; mean [range] age, 25.9 [19-40] years) were recruited in the study. Fifteen patients (71%; 95% CI, 48%-89%) developed migraine attacks after VIP compared with 1 patient (5%; 95% CI, 0%-24%) who developed a migraine attack after placebo (P < .001). The VIP-induced migraine attacks mimicked patients' spontaneous attacks. The area under the curve (AUC) of headache intensity scores (0-12 hours), as well as the AUC of the superficial temporal artery diameter (0-180 minute) were significantly greater after VIP compared with placebo (AUC0-12h, P = .003; AUC0-180min, P < .001). Conclusions and Relevance: A 2-hour infusion of VIP caused migraine attacks, suggesting an important role of VIP in migraine pathophysiology. VIP and its receptors could be potential targets for novel migraine drugs. Trial Registration: ClinicalTrials.gov Identifier: NCT04260035.

Comparative Neurology of Circadian Photoreception: The Retinohypothalamic Tract (RHT) in Sighted and Naturally Blind Mammals.

The mammalian eye contains two systems for light perception: an image detecting system constituted primarily of the classical photoreceptors, rods and cones, and a non-image forming system (NIF) constituted of a small group of intrinsically photosensitive retinal ganglion cells driven by melanopsin (mRGCs). The mRGCs receive input from the outer retina and NIF mediates light entrainment of circadian rhythms, masking behavior, light induced inhibition of nocturnal melatonin secretion, pupillary reflex (PLR), and affect the sleep/wake cycle. This review focuses on the mammalian NIF and its anatomy in the eye as well as its neuronal projection to the brain. This pathway is known as the retinohypothalamic tract (RHT). The development and functions of the NIF as well as the knowledge gained from studying gene modified mice is highlighted. Furthermore, the similarities of the NIF between sighted (nocturnal and diurnal rodent species, monkeys, humans) and naturally blind mammals (blind mole rats Spalax ehrenbergi and the Iberian mole, Talpa occidentalis) are discussed in relation to a changing world where increasing exposure to artificial light at night (ALAN) is becoming a challenge for humans and animals in the modern society.

A Human Cellular Model for Colorectal Anastomotic Repair: The Effect of Localization and Transforming Growth Factor-ß1 Treatment on Collagen Deposition and Biomarkers.

Anastomotic leakage (AL) is a devastating complication after colorectal surgery, possibly due to the loss of stabilizing collagen fibers in the submucosa. Our aim was to assess the formation of collagen in the colon versus the rectum with or without transforming growth factor (TGF)-ß1 exposure in a human cellular model of colorectal repair. Primary fibroblasts were isolated by an explant procedure from clinically resected tissue rings during anastomosis construction in 19 consecutive colorectal patients who underwent laparoscopy. The cells, identified as fibroblasts by morphologic characteristics and flow cytometry analysis (CD90+), were cultured for 8 days and in 12 patients in the presence of 1 ng/mL TGF-ß1. Total collagen deposition was measured colorimetrically after Sirius red staining of fixed cell layers, and type I, III, and VI collagen biosynthesis and degradation were specifically determined by the biomarkers PINP, PRO-C3, PRO-C6, and C3M in conditioned media by competitive enzyme-linked immunosorbent assays. Total collagen deposition by fibroblasts from the colon and rectum did not significantly differ. TGF-ß1 treatment increased PINP, PRO-C6, and total collagen deposition. Mechanistically, TGF-ß1 treatment increased COL1A1 and ACTA2 (encoding α-smooth muscle actin), and decreased COL6A1 and MMP2 mRNA levels in colorectal fibroblasts. In conclusion, we found no effect of anatomic localization on collagen production by fibroblasts derived from the large intestine. TGF-ß1 represents a potential therapeutic agent for the prevention of AL by increasing type I collagen synthesis and collagen deposition.

Early treatment with sumatriptan prevents PACAP38-induced migraine: A randomised clinical trial.

OBJECTIVE: To determine whether early treatment with sumatriptan can prevent PACAP38-induced migraine attacks. METHODS: A total of 37 patients with migraine without aura were enrolled between July 2018 to December 2019. All patients received an intravenous infusion of 10 picomole/kg/min of PACAP38 over 20 min followed by an intravenous infusion of 4 mg sumatriptan or placebo over 10 min on two study days in a randomised, double-blind, placebo-controlled, crossover study. RESULTS: Of 37 patients enrolled, 26 (70.3%) completed the study and were included in analyses. Of the 26 patients, four (15%) developed a PACAP38-induced migraine attack on sumatriptan and 11 patients (42%) on placebo (p = 0.016). There were no differences in area under the curve for headache intensity between sumatriptan (mean AUC 532) and placebo (mean AUC 779) (p = 0.35). Sumatriptan significantly constricted the PACAP38-dilated superficial temporal artery immediately after infusion (T30) compared with infusion of placebo (p < 0.001).Conclusions and relevance: Early treatment with intravenously administered sumatriptan prevented PACAP38-induced migraine. Prevention of migraine attacks was associated with vasoconstriction by sumatriptan in the earliest phases of PACAP provocation. These results suggest that sumatriptan prevents PACAP38-induced migraine by modulation of nociceptive transmission within the trigeminovascular system.Trial Registration: ClinicalTrials.gov (NCT03881644).