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Saema Ansar and Lars Edvinsson

Object

Cerebral ischemia remains the key cause of disability and death in the late phase after subarachnoid hemorrhage (SAH), and its pathogenesis is still poorly understood. The purpose of this study was to examine whether the change in intracranial pressure or the extravasated blood causes the late cerebral ischemia and the upregulation of receptors or the cerebral vasoconstriction observed following SAH.

Methods

Rats were allocated to 1 of 3 experimental conditions: 1) cisternal injection of 250 μl blood (SAH Group), 2) cisternal injection of 250 μl NaCl (Saline Group), or 3) the same procedure but without fluid injection (Sham Group). Two days after the procedure, the basilar and middle cerebral arteries were harvested, and contractile responses to endothelin (ET)–1 and 5-carboxamidotryptamine (5-CT) were investigated by means of myography. In addition, real-time polymerase chain reaction was used to determine the mRNA levels for ETA, ETB, and 5-HT1 receptors. Regional and global cerebral blood flow (CBF) were quantified by means of an autoradiographic technique.

Results

Compared with the sham condition, both SAH and saline injection resulted in significantly enhanced contraction of cerebral arteries in response to ET-1 and 5-CT. Regional and global CBF were reduced both in the Saline and SAH groups compared with the Sham Group. The mRNA levels for ETB and 5-HT1B receptors were upregulated after SAH and saline injection compared with the sham procedure. The effects in all parameters were more pronounced for SAH than for saline injection.

Conclusions

This study revealed that both the elevation of intracranial pressure and subarachnoid blood per se contribute approximately equally to the late CBF reductions and receptor upregulation following SAH.

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Fredric B. Meyer

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Petter Vikman, Saema Ansar and Lars Edvinsson

Object

Subarachnoid hemorrhage (SAH) results in the expression of inflammatory and extracellular matrix (ECM)–related genes and various G protein–coupled receptors. In the present study, the authors evaluated the time course and sequence of the transduction pathways, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase–1 and 2 (ERK1/2), and associated transcription factor activation as well as gene regulation and associated protein levels.

Methods

Subarachnoid hemorrhage was induced in rats by injecting 250 μl of blood into the suprachiasmatic cistern, and gene regulation in the cerebral arteries was examined at various points in time following SAH by using quantitative polymerase chain reaction (PCR) and immunohistochemistry.

Results

Immunohistochemical findings demonstrated that SAH phosphorylates and activates p38 and ERK1/2 as well as the downstream transcription factors Elk-1 and activating transcription factor–2. The pattern of activation consists of a rapid phase within the first few hours and a late phase that occurs from 24 to 48 hours. Activation is followed by an increase in the transcription of the inflammatory and ECM-related genes (IL6, TNFα, IL1β, CXCL1, CXCL2, CCL20, MMP8, MMP9, MMP13, and iNOS), as demonstrated using real-time PCR. For MMP13 and iNOS, the changes in transcription were translated into functional proteins, as revealed on immunohistochemistry.

Conclusions

Activation of the p38 and ERK1/2 signaling pathways and their downstream transcription factors can explain the increase in the transcription of the genes studied. This increase and the subsequent augmentation in protein levels suggest that the inflammatory response may in part explain the remodeling that occurs in cerebral arteries following SAH.

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Saema Ansar, Niels-Aage Svendgaard and Lars Edvinsson

Object

Cerebral vasospasm following subarachnoid hemorrhage (SAH) leads to reduced cerebral blood flow (CBF) and to cerebral ischemia, in some cases even producing infarction and long-term disability. The goal of the present study was to investigate the hypothesis that inhibition of neurokinin-1 receptors (NK1Rs) by administration of L-822429 blunts the decrease in CBF as well as cerebrovascular receptor upregulation in an animal model of SAH.

Methods

Subarachnoid hemorrhage was induced in rats by injection of 250 μl of blood into the prechiasmatic cistern. The NK1R inhibitor L-822429 was injected intracisternally 30 minutes and 24 hours after the induction of SAH. Two days after SAH induction, the basilar arteries were harvested, and contractile responses to endothelin-1 (ET-1, an ETA- and ETB-receptor agonist) and 5-carboxamidotryptamine (a 5-hydroxytryptamine-1 [5-HT1]-receptor agonist) were investigated using sensitive myographs. To determine whether NK1R inhibition had an influence on local CBF after post-SAH, a quantitative autoradiographic technique was used.

After SAH, the vascular receptor phenotype was changed in cerebral arteries through upregulation of contractile ETB and 5-HT1B receptors, while regional and total CBF were markedly reduced. Treatment with the selective NK1R inhibitor L-822429 prevented both the receptor upregulation and the reduction in regional and global CBF.

Conclusions

The data reveal the coregulation of vascular receptor changes and blood flow effects, and also show that interaction with a small-molecule NK1R antagonist is a promising area of focus for the development of specific treatments for SAH.

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Lars Edvinsson, Kai C. Nielsen, Christer Owman and Kurt A. West

✓ Ventricular fluid pressure was measured continuously for 50 hours in conscious rabbits via a cannula implanted into the left lateral ventricle. Intraventricular injection of 25 µg reserpine (in a volume of 10 µl) resulted in increased pressure compared to that in non-injected controls during the first 7 to 10 hours. This was interpreted as depletion of noradrenaline in intracranial sympathetic nerves leading to increased cerebral blood volume and increased cerebrospinal fluid production. During the remainder of the experiment, the ventricular fluid pressure was reduced, probably due to a predominance of the central depressant effects of reserpine.

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Carl Christian Larsen, Gro Klitgaard Povlsen, Marianne Nelly Paola Rasmussen and Lars Edvinsson

Object

Delayed cerebral ischemia after subarachnoid hemorrhage (SAH) remains a major cause of death and disability. It has been hypothesized that cerebrovascular upregulation of vasoconstrictor receptors is a key step in the development of delayed cerebral ischemia. Upregulation of endothelin-B (ETB) and 5-hydroxytryptamine 1B (5-HT1B) receptors has been demonstrated in cerebral artery smooth muscles in the delayed ischemic phase after experimental SAH, and intracellular signaling via the mitogen-activated protein kinase kinase (MEK)–extracellular signal-regulated kinase 1/2 pathway has been shown to be involved in this upregulation. The aim in the present study was to determine whether treatment with the MEK1/2 inhibitor U0126 can prevent cerebrovascular receptor upregulation and improve functional outcome after experimental SAH in rats.

Methods

Subarachnoid hemorrhage was induced in male Sprague-Dawley rats by the injection of 250 μl of autologous blood into the basal cisterns. Either U0126 or vehicle was intracisternally administered at 6, 12, 24, and 36 hours after SAH. Smooth muscle ETB and 5-HT1B receptor upregulation was studied in isolated cerebral artery segments through immunohistochemical and myographic studies of contractile responses to receptor-specific agonists. Gross sensorimotor function in the rats after SAH was assessed using a rotating pole test.

Results

Contractile concentration-response curves for middle cerebral artery (MCA) and basilar artery (BA) segments to endothelin-1 (ET-1) and 5-carboxamidotryptamine (5-CT) were shifted leftward for SAH-induced compared with shamoperated rats due to enhanced contractile responses to individual doses of the agonists (for example, contractile responses of the BA to 3 × 10−10 M of ET-1 and 3 × 10−7 M of 5-CT were 9.98 ± 5.01% and 16.75 ± 3.62% of the maximal contractile capacity, respectively, in sham-operated rats and 62.78 ± 9.9% and 45.44 ± 10.62%, respectively, in SAH-induced rats). In vivo treatment with 0.19 μg/kg U0126 normalized responses in the SAH-induced rats to levels in the sham-operated rats. Protein expression of ETB and 5-HT1B receptors in cerebrovascular smooth muscles from SAH-induced rats was increased to 175 ± 33.17% and 167.7 ± 24.74%, respectively, of the levels in sham-operated rats. Endothelin-B and 5-HT1B expression levels in U0126-treated SAH-induced rats were at the levels in sham-operated rats (101.9 ± 13.38% and 91.44 ± 16.75%, respectively). In a rotating pole test used to assess gross sensorimotor function on the 2nd day after surgery, sham-operated rats achieved an average score of 5.37 ± 0.23, SAH-induced rats scored 3.35 ± 0.67, and SAH-induced U0126-treated rats scored 5.00 ± 0.4.

Conclusions

The authors demonstrated that experimental SAH induces upregulation of ETB and 5-HT1B receptors in cerebrovascular smooth muscles and that treatment with the MEK1/2 inhibitor U0126 abolishes this receptor upregulation. They also demonstrated that experimental SAH results in sensorimotor deficits as assessed by a rotating pole test. These deficits were alleviated by U0126 treatment, suggesting that cerebrovascular receptor upregulation is critical for the functional outcome of delayed cerebral ischemia. The authors suggest that inhibition of MEK1/2 may be a promising new SAH treatment strategy.

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Petter Vikman, Saema Beg, Tejvir Khurana, Jacob Hansen-Schwartz and Lars Edvinsson

Object

The authors investigated early changes in the cerebral arteries of rats that occur after subarachnoid hemorrhage (SAH).

Methods

Messenger RNA was investigated by performing microarray and quantitative real-time polymerase chain reaction (PCR) analyses, and protein expression was shown by performing immunohistochemical studies. The array data indicated that the initial processes that occur after SAH involve activation of genes involved in angiogenesis, inflammation, and extracellular matrix (ECM) remodeling. The real-time PCR investigation confirmed upregulation of genes that were observed using the microarray to be regulated, including iNOS, MMP13, and cxcl2. The authors also verified the upregulation of previously implicated genes for G-protein–coupled receptors (endothelin B [ETB], angiotensin 1 [AT1], and AT2) and metalloproteinase 9. The results of an immunohistochemical study confirmed that receptor genes that were seen to be regulated produced an increase in protein expression. Double immunostaining of rat cerebral arteries with endothelial cell– or smooth-muscle cell–specific antibodies verified that an increase in ETB, 5-hydrotryptamine (5-HT1B), and 5-HT1D receptor expression occurs in smooth-muscle cells.

Conclusions

Processes occurring after SAH lead to enhanced arterial contractility and ECM remodeling either directly or through angiogenesis and inflammation. These processes are active via an increase in metalloproteinase expression, the presence of proangiogenic factors, and the expression of proinflammatory genes.

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Tia Juana Delgado-Zygmunt, Mohammed Abdul-Rahman Arbab, Lars Edvinsson, Inger Jansen and Niels Aage Svendgaard

✓ Cisternal blood injection in the rat induces a biphasic angiographic vasospasm, with a maximal acute spasm at 10 minutes and a maximal late spasm at 2 days after the subarachnoid hemorrhage (SAH). Depletion of substance P-containing sensory nerves to the cerebral arteries with capsaicin prior to SAH prevents the development of both acute and late spasm. Intrathecal administration of the substance P antagonist spantide 2 hours prior to SAH also prevents the development of vasospasm, while spantide administration 1 hour before SAH only hinders the occurrence of late vasospasm. Intracisternal administration of spantide 2 hours post-SAH prevents the development of late vasospasm. This antagonist per se can induce a short-lasting dose-dependent angiographic vasoconstriction. Substance P-containing nerve fibers on the cerebral arteries could constitute the sensory link in a reflex arc system involved in the development of vasospasm in which the presence of blood in the subarachnoid space stimulates sensory substance P-containing nerve fibers on the cerebral arteries inducing a centripetal impulse to the A2-nucleus tractus solitarius and setting into motion the events in the brain stem leading to acute and late vasospasm.

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Jacob Hansen-Schwartz, Natalie Løvland Hoel, Cang-Bao Xu, Niels-Aage Svendgaard and Lars Edvinsson

Object. Cerebral vasospasm following subarachnoid hemorrhage (SAH) leads to reduced blood flow in the brain. Inspired by organ culture—induced changes in the receptor phenotype of cerebral arteries, the authors investigated possible changes in the 5-hydroxytryptamine (HT) receptor phenotype after experimental SAH.

Methods. Experimental SAH was induced in rats by using an autologous prechiasmatic injection of arterial blood. Two days later, the middle cerebral artery (MCA), posterior communicating artery (PCoA), and basilar artery (BA) were harvested and examined functionally with the aid of a sensitive in vitro pharmacological method and molecularly by performing quantitative real-time reverse transcription—polymerase chain reaction (PCR).

In the MCA and BA the 5-HT1B receptor was upregulated, as determined through both functional and molecular analysis. In response to selective 5-HT1 receptor agonists both the negative logarithm of the 50% effective concentration was increased (one log unit in the MCA and one half unit in the BA), as was the agonist's potency (increased by 50% in the MCA and doubled in the BA). In addition, the authors found an approximately fourfold increase in the number of copies of messenger RNA coding for the 5-HT1B receptor as determined by quantitative real-time PCR. In the PCoA no upregulation of the 5-HT1B receptor was observed.

Conclusions. Changes in the receptor phenotype in favor of contractile receptors may well represent the end stage in a sequence of events leading from SAH to the actual development of cerebral vasospasm. Insight into the mechanism of upregulation may provide new targets for developing specific treatment against cerebral vasospasm.