Sara Ellinor Johansson

Localization of CGRP, CGRP receptor, PACAP and glutamate in trigeminal ganglion. Relation to the blood-brain barrier.

Calcitonin gene-related peptide (CGRP) receptor antagonists have demonstrated anti-migraine efficacy. One remaining question is where do these blockers act? We hypothesized that the trigeminal ganglion could be one possible site. We examined the binding sites of a CGRP receptor antagonist (MK-3207) and related this to the expression of CGRP and its receptor in rhesus trigeminal ganglion. Pituitary adenylate cyclase-activating polypeptide (PACAP) and glutamate were examined and related to the CGRP system. Furthermore, we examined if the trigeminal ganglion is protected by the blood-brain barrier (BBB). Autoradiography was performed with [(3)H]MK-3207 to demonstrate receptor binding sites in rhesus trigeminal ganglion (TG). Immunofluorescence was used to correlate binding and the presence of CGRP and its receptor components, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), and the distribution of PACAP and glutamate in rhesus and rat TG. Evans blue was used to examine large molecule penetration into the rat TG. High receptor binding densities were found in rhesus TG. Immunofluorescence revealed expression of CGRP, CLR and RAMP1 in trigeminal cells. CGRP positive neurons expressed PACAP but not glutamate. Some neurons expressing CLR and RAMP1 co-localized with glutamate. Evans blue revealed that the TG is not protected by BBB. This study demonstrates CGRP receptor binding sites and expression of the CGRP receptor in rhesus and rat TG. The expression pattern of PACAP and glutamate suggests a possible interaction between the glutamatergic and CGRP system. In rat the TG is outside the BBB, suggesting that molecules do not need to be CNS-penetrant to block these receptors.

Expressional Changes in Cerebrovascular Receptors after Experimental Transient Forebrain Ischemia

Background Global ischemic stroke is one of the most prominent consequences of cardiac arrest, since the diminished blood flow to the brain results in cell damage and sometimes permanently impaired neurological function. The post-arrest period is often characterised by cerebral hypoperfusion due to subacute hemodynamic disturbances, the pathophysiology of which are poorly understood. In two other types of stroke, focal ischemic stroke and subarachnoid hemorrhage, it has earlier been demonstrated that the expression of certain vasoconstrictor receptors is increased in cerebral arteries several days after the insult, a phenomenon that leads to increased contraction of cerebral arteries, reduced perfusion of the affected area and worsened ischemic damage. Based on these findings, the aim of the present study was to investigate if transient global cerebral ischemia is associated with upregulation of vasoconstrictive endothelin and 5-hydroxytryptamine receptors in cerebral arteries. Experimental transient forebrain ischemia of varying durations was induced in male wistar rats, followed by reperfusion for 48 hours. Neurological function was assessed daily by three different tests and cerebrovascular expression and contractile function of endothelin and 5-hydroxytryptamine receptors were evaluated by wire myography, immunohistochemistry and western blotting. Results Transient forebrain ischemia induced neurological deficits as well as functional upregulation of vasoconstrictive ETB and 5-HT1B receptors in cerebral arteries supplying mid- and forebrain regions. No receptor upregulation was seen in arteries supplying the hindbrain. Immunohistochemical stainings and western blotting demonstrated expressional upregulation of these receptor subtypes in the mid- and forebrain arteries and confirmed that the receptors were located in the smooth muscle layer of the cerebral arteries. Conclusions This study reveals a new pathophysiological aspect of global ischemic stroke, namely expressional upregulation of vasoconstrictor receptors in cerebral arteries two days after the insult, which might contribute to cerebral hypoperfusion and delayed neuronal damage after cardiac arrest.

Low breast milk levels of long-chain n-3 fatty acids in allergic women, despite frequent fish intake

Cite this as: S. Johansson, A. E. Wold and A‐S Sandberg, Clinical & Experimental Allergy, 2011 (41) 505–515.

Long-chain polyunsaturated fatty acids are consumed during allergic inflammation and affect T helper type 1 (Th1)- and Th2-mediated hypersensitivity differently

Studies have shown that atopic individuals have decreased serum levels of n‐3 fatty acids. Indicating these compounds may have a protective effect against allergic reaction and/or are consumed during inflammation. This study investigated whether fish (n‐3) or sunflower (n‐6) oil supplementation affected T helper type 1 (Th1)‐ and Th2‐mediated hypersensitivity in the skin and airways, respectively, and whether the fatty acid serum profile changed during the inflammatory response. Mice were fed regular chow, chow + 10% fish oil or chow + 10% sunflower oil. Mice were immunized with ovalbumin (OVA) resolved in Th1 or Th2 adjuvant. For Th1 hypersensitivity, mice were challenged with OVA in the footpad. Footpad swelling, OVA‐induced lymphocyte proliferation and cytokine production in the draining lymph node were evaluated. In the airway hypersensitivity model (Th2), mice were challenged intranasally with OVA and the resulting serum immunoglobulin (Ig)E and eosinophilic lung infiltration were measured. In the Th1 model, OVA‐specific T cells proliferated less and produced less interferon (IFN)‐γ, tumour necrosis factor (TNF) and interleukin (IL)‐6 in fish oil‐fed mice versus controls. Footpad swelling was reduced marginally. In contrast, mice fed fish oil in the Th2 model produced more OVA‐specific IgE and had slightly higher proportions of eosinophils in lung infiltrate. A significant fall in serum levels of long‐chain n‐3 fatty acids accompanied challenge and Th2‐mediated inflammation in Th2 model. Fish oil supplementation affects Th1 and Th2 immune responses conversely; significant consumption of n‐3 fatty acids occurs during Th2‐driven inflammation. The latter observation may explain the association between Th2‐mediated inflammation and low serum levels of n‐3 fatty acids.

High Levels of Both n-3 and n-6 Long-Chain Polyunsaturated Fatty Acids in Cord Serum Phospholipids Predict Allergy Development

Background Long-chain polyunsaturated fatty acids (LCPUFAs) reduce T-cell activation and dampen inflammation. They might thereby counteract the neonatal immune activation and hamper normal tolerance development to harmless environmental antigens. We investigated whether fatty acid composition of cord serum phospholipids affects allergy development up to age 13 years. Methods From a population-based birth-cohort born in 1996/7 and followed until 13 years of age (n = 794), we selected cases with atopic eczema (n = 37) or respiratory allergy (n = 44), as well as non-allergic non-sensitized controls (n = 48) based on diagnosis at 13 years of age. Cord and maternal sera obtained at delivery from cases and controls were analysed for proportions of saturated, monounsaturated and polyunsaturated fatty acids among serum phospholipids. Results The cord serum phospholipids from subject who later developed either respiratory allergy or atopic eczema had significantly higher proportions of 5/8 LCPUFA species, as well as total n-3 LCPUFA, total n-6 LCPUFA and total LCPUFA compared to cord serum phospholipids from controls who did not develop allergy (P<0.001 for all comparisons). Conversely, individuals later developing allergy had lower proportion of the monounsaturated fatty acid 18∶1n-9 as well as total MUFA (p<0.001) among cord serum phospholipids. The risk of respiratory allergy at age 13 increased linearly with the proportion of n-3 LCPUFA (Ptrend<0.001), n-6 LCPUFA (Ptrend = 0.001), and total LCPUFA (Ptrend<0.001) and decreased linearly with the proportions of total MUFA (Ptrend = 0.025) in cord serum phospholipids. Furthermore, Kaplan-Meier estimates of allergy development demonstrated that total LCPUFA proportion in cord serum phospholipids was significantly associated with respiratory allergy (P = 0.008) and sensitization (P = 0.002), after control for sex and parental allergy. Conclusion A high proportion of long-chain PUFAs among cord serum phospholipids may predispose to allergy development. The mechanism is unknown, but may involve dampening of the physiologic immune activation in infancy needed for proper maturation of the infants immune system.

Early events triggering delayed vasoconstrictor receptor upregulation and cerebral ischemia after subarachnoid hemorrhage

BackgroundUpregulation of vasoconstrictor receptors in cerebral arteries, including endothelin B (ETB) and 5-hydroxytryptamine 1B (5-HT1B) receptors, has been suggested to contribute to delayed cerebral ischemia, a feared complication after subarachnoid hemorrhage (SAH). This receptor upregulation has been shown to be mediated by intracellular signalling via the mitogen activated protein kinase kinase (MEK1/2) - extracellular regulated kinase 1/2 (ERK1/2) pathway. However, it is not known what event(s) that trigger MEK-ERK1/2 activation and vasoconstrictor receptor upregulation after SAH.We hypothesise that the drop in cerebral blood flow (CBF) and wall tension experienced by cerebral arteries in acute SAH is a key triggering event. We here investigate the importance of the duration of this acute CBF drop in a rat SAH model in which a fixed amount of blood is injected into the prechiasmatic cistern either at a high rate resulting in a short acute CBF drop or at a slower rate resulting in a prolonged acute CBF drop.ResultsWe demonstrate that the duration of the acute CBF drop is determining for a) degree of early ERK1/2 activation in cerebral arteries, b) delayed upregulation of vasoconstrictor receptors in cerebral arteries and c) delayed CBF reduction, neurological deficits and mortality. Moreover, treatment with an inhibitor of MEK-ERK1/2 signalling during an early time window from 6 to 24 h after SAH was sufficient to completely prevent delayed vasoconstrictor receptor upregulation and improve neurological outcome several days after the SAH.ConclusionsOur findings suggest a series of events where 1) the acute CBF drop triggers early MEK-ERK1/2 activation, which 2) triggers the transcriptional upregulation of vasoconstrictor receptors in cerebral arteries during the following days, where 3) the resulting enhanced cerebrovascular contractility contribute to delayed cerebral ischemia.

Cerebrovascular endothelin-1 hyper-reactivity is associated with transient receptor potential canonical channels 1 and 6 activation and delayed cerebral hypoperfusion after forebrain ischaemia in rats

In this study, we aimed to investigate whether changes in cerebrovascular voltage‐dependent calcium channels and non‐selective cation channels contribute to the enhanced endothelin‐1‐mediated vasoconstriction in the delayed hypoperfusion phase after experimental transient forebrain ischaemia.

Early MEK1/2 Inhibition after Global Cerebral Ischemia in Rats Reduces Brain Damage and Improves Outcome by Preventing Delayed Vasoconstrictor Receptor Upregulation

Background Global cerebral ischemia following cardiac arrest is associated with increased cerebral vasoconstriction and decreased cerebral blood flow, contributing to delayed neuronal cell death and neurological detriments in affected patients. We hypothesize that upregulation of contractile ETB and 5-HT1B receptors, previously demonstrated in cerebral arteries after experimental global ischemia, are a key mechanism behind insufficient perfusion of the post-ischemic brain, proposing blockade of this receptor upregulation as a novel target for prevention of cerebral hypoperfusion and delayed neuronal cell death after global cerebral ischemia. The aim was to characterize the time-course of receptor upregulation and associated neuronal damage after global ischemia and investigate whether treatment with the MEK1/2 inhibitor U0126 can prevent cerebrovascular receptor upregulation and thereby improve functional outcome after global cerebral ischemia. Incomplete global cerebral ischemia was induced in Wistar rats and the time-course of enhanced contractile responses and the effect of U0126 in cerebral arteries were studied by wire myography and the neuronal cell death by TUNEL. The expression of ETB and 5-HT1B receptors was determined by immunofluorescence. Results Enhanced vasoconstriction peaked in fore- and midbrain arteries 3 days after ischemia. Neuronal cell death appeared initially in the hippocampus 3 days after ischemia and gradually increased until 7 days post-ischemia. Treatment with U0126 normalised cerebrovascular ETB and 5-HT1B receptor expression and contractile function, reduced hippocampal cell death and improved survival rate compared to vehicle treated animals. Conclusions Excessive cerebrovascular expression of contractile ETB and 5-HT1B receptors is a delayed response to global cerebral ischemia peaking 3 days after the insult, which likely contributes to the development of delayed neuronal damage. The enhanced cerebrovascular contractility can be prevented by treatment with the MEK1/2 inhibitor U0126, diminishes neuronal damage and improves survival rate, suggesting MEK1/2 inhibition as a novel strategy for early treatment of neurological consequences following global cerebral ischemia.

Enhanced Endothelin-1 Mediated Vasoconstriction of the Ophthalmic Artery May Exacerbate Retinal Damage after Transient Global Cerebral Ischemia in Rat

Cerebral vasculature is often the target of stroke studies. However, the vasculature supplying the eye might also be affected by ischemia. The aim of the present study was to investigate if the transient global cerebral ischemia (GCI) enhances vascular effect of endothelin-1 (ET-1) and 5-hydroxytryptamine/serotonin (5-HT) on the ophthalmic artery in rats, leading to delayed retinal damage. This was preformed using myography on the ophthalmic artery, coupled with immunohistochemistry and electroretinogram (ERG) to assess the ischemic consequences on the retina. Results showed a significant increase of ET-1 mediated vasoconstriction at 48 hours post ischemia. The retina did not exhibit any morphological changes throughout the study. However, we found an increase of GFAP and vimentin expression at 72 hours and 7 days after ischemia, indicating Müller cell mediated gliosis. ERG revealed significantly decreased function at 72 hours, but recovered almost completely after 7 days. In conclusion, we propose that the increased contractile response via ET-1 receptors in the ophthalmic artery after 48 hours may elicit negative retinal consequences due to a second ischemic period. This may exacerbate retinal damage after ischemia as illustrated by the decreased retinal function and Müller cell activation. The ophthalmic artery and ET-1 mediated vasoconstriction may be a valid and novel therapeutic target after longer periods of ischemic insults.

Vascular and molecular pharmacology of the metabolically stable CGRP analogue, SAX

&NA; The main purpose of this study was to compare in vitro pharmacological properties of human &agr;CGRP (CGRP) and a recently discovered metabolically stable CGRP analogue, SAX, in isolated rat and human artery segments. In rat, CGRP and SAX induced similar vasodilatory responses in isolated mesenteric artery with the potency of SAX being lower than that of CGRP (vasodilatory pEC50 8.2 ± 0.12 and 9.0 ± 0.11, respectively). A corresponding difference in receptor binding affinity of SAX and CGRP was determined in rat cerebral membranes (pKi 8.3 ± 0.19 and 9.3 ± 0.14, respectively). CGRP and SAX‐induced vasodilation was antagonised with similar potencies by the CGRP receptor antagonist BIBN4096BS supporting a uniform receptor population for the agonists. In human tissue, SAX and CGRP induced similar pharmacological responses with different potencies in subcutaneous artery (vasodilatory pEC50 8.8 ± 0.18 and 9.5 ± 0.13, respectively) and human recombinant receptors (cAMP signalling pEC50 9.1 ± 0.16 and 10.2 ± 0.19). Like in the rat mesenteric artery, both SAX and CGRP‐responses were inhibited by the CGRP receptor antagonist BIBN4096BS with similar antagonistic potencies. In conclusion, all pharmacological characteristics of SAX and CGRP in human and rat sources points towards action via a uniform BIBN4096BS sensitive receptor population with the potency of SAX being 5–10 fold lower than that of CGRP. Graphical abstract Figure. No Caption available.