| 1. |
- Blixt, Frank W., et al.
(författare)
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Distribution of CGRP and its receptor components CLR and RAMP1 in the rat retina
- 2017
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Ingår i: Experimental Eye Research. - : Elsevier BV. - 0014-4835. ; 161, s. 124-131
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Tidskriftsartikel (refereegranskat)abstract
- Calcitonin gene-related peptide (CGRP) is a 37 amino acid neuropeptide with several functions including vasodilation, the perception of painful stimuli, and inflammation. The CGRP receptor consists of two main components; calcitonin-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). While there is a growing recognition that CGRP plays a key role in migraine, the function of CGRP in the retina has not been fully established. This study aims to investigate the distribution of CGRP and its two receptor components in the rat retina, visually by immunohistochemistry and quantitatively using flow cytometry. CGRP immunoreactivity was found in the Müller cells while CLR/RAMP1 was located in the nerve fiber layer. Furthermore, since almost all RAMP1 immunoreactive cells co-express CLR, we propose that RAMP1 expression in the retina reflects functional CGRP receptors.
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| 2. |
- Christensen, Simon T., et al.
(författare)
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MEK1/2 inhibitor U0126, but not nimodipine, reduces upregulation of cerebrovascular contractile receptors after subarachnoid haemorrhage in rats
- 2019
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 14:4
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Tidskriftsartikel (refereegranskat)abstract
- Vascular pathophysiological changes after haemorrhagic stroke, such as phenotypic modulation of the cerebral arteries and cerebral vasospasms, are associated with delayed cerebral ischemia (DCI) and poor outcome. The only currently approved drug treatment shown to reduce the risk of DCI and improve neurologic outcome after aneurysmal subarachnoid haemorrhage (SAH) is nimodipine, a dihydropyridine L-type voltage-gated Ca 2+ channel blocker. MEK1/2 mediated transcriptional upregulation of contractile receptors, including endothelin-1 (ET-1) receptors, has previously been shown to be a factor in the pathology of SAH. The aim of the study was to compare intrathecal and subcutaneous treatment regimens of nimodipine and intrathecal treatment regimens of U0126, a MEK1/2 inhibitor, in a single injection experimental rat SAH model with post 48 h endpoints consisting of wire myography of cerebral arteries, flow cytometry of cerebral arterial tissue and behavioural evaluation. Following ET-1 concentration-response curves, U0126 exposed arteries had a significantly lower ET-1 max than vehicle arteries. Arteries from both the intrathecal- and subcutaneous nimodipine treated animals had significantly higher ET-1 max contractions than the U0126 arteries. Furthermore, Ca 2+ concentration response curves (precontracted with ET-1 and in the presence of nimodipine) showed that nimodipine treatment could result in larger nimodipine insensitive contractions compared to U0126. Flow cytometry showed decreased protein expression of the ET B receptor in U0126 treated cerebral vascular smooth muscle cells compared to vehicle. Only U0126 treatment lowered ET-1 max contractions and ET B receptor levels, as well as decreased the contractions involving nimodipine-insensitive Ca 2+ channels, when compared to both intrathecal and subcutaneous nimodipine treatment. This indicate that targeting gene expression might be a better strategy than blocking specific receptors or ion channels in future treatments of SAH.
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| 3. |
- Erdling, André, et al.
(författare)
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Changes in P2Y6 receptor-mediated vasoreactivity following focal and global ischemia
- 2022
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Ingår i: Physiological Reports. - : Wiley. - 2051-817X. ; 10:8
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Tidskriftsartikel (refereegranskat)abstract
- Ischemia, both in the form of focal thromboembolic stroke and following subarachnoid hemorrhage (SAH), causes upregulation of vasoconstrictive receptor systems within the cerebral vasculature. Descriptions regarding changes in purinergic signaling following ischemia are lacking, especially when the importance of purinergic signaling in regulating vascular tone is taken into consideration. This prompted us to evaluate changes in P2Y6-mediated vasomotor reactivity in two different stroke models in rat. We used wire myography to measure changes in cerebral vasoreactivity to the P2Y6 agonist UDP-β-S following either experimental SAH or transient middle cerebral artery occlusion. Changes in receptor localization or receptor expression were evaluated using immunohistochemistry and quantitative flow cytometry. Transient middle cerebral artery occlusion caused an increase in Emax when compared to sham (233.6 [206.1–258.5]% vs. 161.1 [147.1–242.6]%, p = 0.0365). No such change was seen following SAH. Both stroke models were associated with increased levels of P2Y6 receptor expression in the vascular smooth muscle cells (90.94 [86.99–99.15]% and 93.79 [89.96–96.39]% vs. 80.31 [70.80–80.86]%, p = 0.021) and p = 0.039 respectively. There was no change in receptor localization in either of the stroke models. Based on these findings, we conclude that focal ischemic stroke increases vascular sensitivity to UDP-β-S by upregulating P2Y6 receptors on vascular smooth muscle cells while experimental SAH did not induce changes in vasoreactivity in spite of increased P2Y6 receptor expression.
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| 4. |
- Radziwon-Balicka, Aneta, et al.
(författare)
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A novel multicolor flow-cytometry application for quantitative detection of receptors on vascular smooth muscle cells
- 2017
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:10
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Tidskriftsartikel (refereegranskat)abstract
- There is a need to develop new techniques for quantitative measurement of receptors expression on particular vasculature cells types. Here, we describe and demonstrate a novel method to measure quantitatively and simultaneously the expression of endothelin B receptor (ETB) on vascular smooth muscle cells (VSMC). We isolated cells from male rat tissues such as: brain pial, brain intraparenchymal and retina vessels. To analyze solid tissues, a single-cell suspension was prepared by a combined mechanic and enzymatic process. The cells were stained with Fixable Viability Dye, followed by fixation, permeabilization and antibodies staining. The expression of ETB receptors on VSMC was measured by flow-cytometry and visualized by fluorescence microscopy. We obtained a high percentage of viable cells 87.6% ± 1.5% pial; 84.6% ± 4.3% parenchymal and 90.6% ± 4% retina after isolation of single cells. We performed a quantitative measurement of ETB receptor expression on VSMC and we identified two subpopulations of VSMC based on their expression of smooth muscle cells marker SM22α. The results obtained from pial vessels are statistically significant (38.4% ± 4% vs 9.8% ± 3.32%) between the two subpopulations of VSMC. The results obtained from intraparenchymal and retina vessels were not statistically significant. By specific gating on two subpopulations, we were able to quantify the expression of ETB receptors. The two subpopulation expressed the same level of ETB receptor (p = 0.45; p = 0.3; p = 0.42) in pial, parenchymal and retina vessels, respectively. We applied our method to the animals after induction of subarachnoid hemorrhage (SAH). There was statistically significant expression of ETB receptor (p = 0.02) on VSMC between sham 61.4% ± 4% and SAH 77.4% ± 4% rats pial vessels. The presented technique is able to quantitatively and selectively measure the level of protein expression on VSMC. The entire technique is optimized for rat tissue; however the protocol can also be adapted for other species.
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| 5. |
- Spray, Stine, et al.
(författare)
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Alterations in the Cerebral Microvascular Proteome Expression Profile After Transient Global Cerebral Ischemia in Rat
- 2017
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Ingår i: Journal of Molecular Neuroscience. - : Springer Science and Business Media LLC. - 0895-8696 .- 1559-1166. ; 61:3, s. 396-411
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Tidskriftsartikel (refereegranskat)abstract
- This study aimed at obtaining an in-depth mapping of expressional changes of the cerebral microvasculature after transient global cerebral ischemia (GCI) and the impact on these GCI-induced expressional changes of post-GCI treatment with a mitogen-activated protein kinase kinase (MEK1/2) inhibitor. GCI was induced in male Wistar rats followed by treatment with either vehicle or the MEK1/2 inhibitor U0126 every 12 h post-GCI. Seventy-two hours after GCI or sham surgery, the cerebral microvasculature was isolated and the protein content analysed with state-of-the-art mass spectrometry. The proteomic profile of the isolated cerebral microvasculature 72 h after GCI (compared to sham) indicated that the main expressional changes could be divided into nine categories: (1) cellular respiration, (2) remodelling of the extracellular matrix, (3) decreased contractile phenotype, (4) clathrin-mediated endocytosis, (5) ribosomal activity, (6) expression of chromatin structure-related proteins, (7) altered synaptic activity, (8) altered G-protein signalling and (9) instability of the membrane potential. Treatment with U0126 partly normalized the expression of one or more of the proteins in all nine categories. Flow cytometry confirmed key findings from the proteome such as upregulation of the extracellular proteins lamininβ2 and nidogen2 (p <0.05) after GCI. These results provide valuable molecular insight into the broad and complex expressional changes in the cerebral microvasculature after GCI and the effect of early MEK1/2 inhibitor treatment on these changes.
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| 6. |
- Spray, Stine, et al.
(författare)
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Enhanced contractility of intraparenchymal arterioles after global cerebral ischaemia in rat - new insights into the development of delayed cerebral hypoperfusion
- 2017
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Ingår i: Acta Physiologica. - : Wiley. - 1748-1716 .- 1748-1708. ; 220:4, s. 417-431
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Tidskriftsartikel (refereegranskat)abstract
- Aim: Delayed cerebral hypoperfusion is a secondary complication found in the days after transient global cerebral ischaemia that worsens the ischaemic damage inflicted by the initial transient episode of global cerebral ischaemia. A recent study demonstrated increased cerebral vasoconstriction in the large arteries on the brain surface (pial arteries) after global cerebral ischaemia. However, smaller arterioles inside the brain (parenchymal arterioles) are equally important in the regulation of cerebral blood flow and yet their pathophysiology after global cerebral ischaemia is largely unknown. Therefore, we investigated whether increased contractility occurs in the intraparenchymal arterioles. Methods: Global cerebral ischaemia was induced in male Wistar rats by bilateral common carotid occlusion for 15 min combined with hypovolaemia. Regional cerebral blood flow was determined by quantitative autoradiography. Intraparenchymal arterioles were isolated and pressurized, and concentration-response curves to endothelin-1 with and without the endothelin B receptor-selective antagonist BQ788 was generated. Endothelin B receptor expression was investigated by quantitative flow cytometry and immunohistochemistry. Results: We observed increased endothelin-1-mediated contractility of parenchymal arterioles correlating with reduced cerebral blood flow of the cortex, hippocampus and caudate nucleus 48 h after global cerebral ischaemia. The increased endothelin-1-mediated contractility was abolished by BQ788, and the vascular smooth muscle cell-specific expression of endothelin B receptors was significantly increased after global cerebral ischaemia. Conclusion: Increased endothelin-1-mediated contractility and expression of endothelin B receptors in the intraparenchymal vasculature contributes to the development of delayed cerebral hypoperfusion after global cerebral ischaemia in combination with vascular changes of the pial vasculature.
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