The MEK Inhibitor Trametinib Improves Outcomes following Subarachnoid Haemorrhage in Female Rats.

Aneurysmal subarachnoid haemorrhage (SAH) is a haemorrhagic stroke that causes approximately 5% of all stroke incidents. We have been working on a treatment strategy that targets changes in cerebrovascular contractile receptors, by blocking the MEK/ERK1/2 signalling pathway. Recently, a positive effect of trametinib was found in male rats, but investigations of both sexes in pre-clinical studies are an important necessity. In the current study, a SAH was induced in female rats, by autologous blood-injection into the pre-chiasmatic cistern. This produces a dramatic, transient increase in intracranial pressure (ICP) and an acute and prolonged decrease in cerebral blood flow. Rats were then treated with either vehicle or three doses of 0.5 mg/kg trametinib (specific MEK/ERK1/2 inhibitor) intraperitoneally at 3, 9, and 24 h after the SAH. The outcome was assessed by a panel of tests, including intracranial pressure (ICP), sensorimotor tests, a neurological outcome score, and myography. We observed a significant difference in arterial contractility and a reduction in subacute increases in ICP when the rats were treated with trametinib. The sensory motor and neurological outcomes in trametinib-treated rats were significantly improved, suggesting that the improved outcome in females is similar to that of males treated with trametinib.

Changes in P2Y6 receptor-mediated vasoreactivity following focal and global ischemia.

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-ß-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-ß-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.

Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model.

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely afflicting a fairly young population. Several animal models of SAH have been developed to investigate the pathophysiological mechanisms behind SAH and to test pharmacological interventions. The pre-chiasmatic, single injection model in the rat presented in this article is an experimental model of SAH with a predetermined blood volume. Briefly, the animal is anesthetized, intubated, and kept under mechanical ventilation. Temperature is regulated with a heating pad. A catheter is placed in the tail artery, enabling continuous blood pressure measurement as well as blood sampling. The atlantooccipital membrane is incised and a catheter for pressure recording is placed in the cisterna magna to enable intracerebral pressure measurement. This catheter can also be used for intrathecal therapeutic interventions. The rat is placed in a stereotaxic frame, a burr hole is drilled anteriorly to the bregma, and a catheter is inserted through the burr hole and placed just anterior to the optic chiasm. Autologous blood (0.3 mL) is withdrawn from the tail catheter and manually injected. This results in a rise of intracerebral pressure and a decrease of cerebral blood flow. The animal is kept sedated for 30 min and given subcutaneous saline and analgesics. The animal is extubated and returned to its cage. The pre-chiasmatic model has a high reproducibility rate and limited variation between animals due to the pre-determined blood volume. It mimics SAH in humans making it a relevant model for SAH research.

Subacute phase of subarachnoid haemorrhage in female rats: Increased intracranial pressure, vascular changes and impaired sensorimotor function.

OBJECTIVE: Early brain injury (EBI) and delayed cerebral ischemia (DCI) after subarachnoid haemorrhage (SAH) has devastating consequences but therapeutic options and the underlying pathogenesis remain poorly understood despite extensive preclinical and clinical research. One of the drawbacks of most preclinical studies to date is that the mechanisms behind DCI after SAH are studied only in male animals. In this study we therefore established a female rat model of SAH in order to determine subacute pathophysiological changes that may contribute to DCI in females. METHODS: Experimental SAH was induced in female rats by intracisternal injection of 300 µL of autologous blood. Sham operation served as a control. Neurological deficits and intracranial pressure measurements were evaluated at both 1 and 2 days after surgery. Additionally, changes in cerebral vascular contractility were evaluated 2 days after surgery using wire myography. RESULTS: SAH in female rats resulted in sensorimotor deficits and decreased general wellbeing on both day 1 and day 2 after SAH. Intracranial pressure uniformly increased in all rats subjected to SAH on day 1. On day 2 the intracranial pressure had increased further, decreased slightly or remained at the level seen on day 1. Furthermore, female rats subjected to SAH developed cortical brain edema. Cerebral arteries, isolated 2 days after SAH, exhibited increased vascular contractions to endothelin-1 and 5-carboxamidotryptamine. CONCLUSION: In the subacute phase after SAH in female rats, we observed increased intracranial pressure, decreased wellbeing, sensorimotor deficits, increased vascular contractility and cortical brain edema. Collectively, these pathophysiological changes may contribute to DCI after SAH in females. Previous studies reported similar pathophysiological changes for male rats in the subacute phase after SAH. Thus, prevention of these gender-independent mechanisms may provide the basis for a universal treatment strategy for DCI after SAH. Nevertheless, preclinical studies of potential therapies should employ both male and female SAH models.

Fremanezumab inhibits vasodilatory effects of CGRP and capsaicin in rat cerebral artery - Potential role in conditions of severe vasoconstriction.

CGRP plays a major role in the pathophysiology of migraine. Concomitant, CGRP plays a role in endogenous neurovascular protection from severe vasoconstriction associated with e.g. cerebral or cardiac ischemia. The CGRP antagonistic antibodies Fremanezumab (TEVA Pharmaceuticals) and Erenumab (Novartis/Amgen) have successfully been developed for the prevention of frequent migraine attacks. Whereas these antibodies might challenge endogenous neurovasular protection during severe cerebral or coronary vasoconstriction, potential future therapeutic CGRP agonists might induce migraine-like headaches in migraineurs. In the current study segments of cerebral artery have been used to obtain mechanistic insight of the CGRP-neutralizing anti-body Fremanezumab in neurovascular regulation in vitro. The basilar artery was selected due to its relevance in subarachnoid hemorrhage (SAH). Erenumab is known to block the human CGRP receptor and Fremanezumab to neutralize both human and rat CGRP. Results confirmed that Erenumab does not block the rat CGRP receptor and that Fremanezumab inhibits the vasodilatory effect induced by both human CGRP, rat CGRP and the metabolically stable CGRP analog, SAX in rat basilar artery. Fremanezumab also inhibits the vasodilatory effect of capsaicin in constricted segments of basilar artery. Capsaicin is used as a pharmacological tool to induce secretion of endogenous perivascular CGRP and our studies confirm that the antibody reach the perivascular sensory synaptic cleft and blocks the vasodilatory response of released CGRP in the present in vitro model. Thus, CGRP neutralization might have the mechanistic potential to block vasoprotective responses to severe vasoconstriction provided they reach the site of action and Fremanezumab is an important tool for future investigations of the impact of CGRP physiology.

Exploration of Physiological and Pathophysiological Implications of miRNA-143 and miRNA-145 in Cerebral Arteries.

Subarachnoid hemorrhage (SAH) is a type of hemorrhagic stroke with a high short-term mortality rate which leads to cognitive impairments that reduce the quality of life of the majority of patients. The miRNA-143/145 cluster is highly expressed in vascular smooth muscle cells (VSMC) and has been shown to be necessary for differentiation and function, as well as an important determinant for phenotypic modulation/switching of VSMCs in response to vascular injury. We aimed to determine whether miRNA-143 and miRNA-145 are important regulators of phenotypical changes of VSMCs in relation to SAH, as well as establishing their physiological role in the cerebral vasculature. We applied quantitative PCR to study ischemia-induced alterations in the expression of miRNA-143 and miRNA-145, for rat cerebral vasculature, in an ex vivo organ culture model and an in vivo SAH model. To determine the physiological importance, we did myograph studies on basilar and femoral arteries from miRNA-143/145 knockout mice. miRNA-143 and miRNA-145 are not upregulated in the vasculature following our SAH model, despite the upregulation of miR-145 in the organ culture model. Regarding physiological function, miRNA-143 and miRNA-145 are very important for general contractility in cerebral vessels in response to depolarization, angiotensin II, and endothelin-1. Applying an anti-miRNA targeting approach in SAH does not seem to be a feasible approach because miRNA-143 and miRNA-145 are not upregulated following SAH. The knockout mouse data suggest that targeting miRNA-143 and miRNA-145 would lead to a general reduced contractility of the cerebral vasculature and unwanted dedifferentiation of VSMCs.

Vascular pathology of large cerebral arteries in experimental subarachnoid hemorrhage: Vasoconstriction, functional CGRP depletion and maintained CGRP sensitivity.

Subarachnoid hemorrhage (SAH) is associated with increased cerebral artery sensitivity to vasoconstrictors and release of the perivascular sensory vasodilator CGRP. In the current study the constrictive phenotype and the vasodilatory effects of exogenous and endogenous perivascular CGRP were characterized in detail applying myograph technology to cerebral artery segments isolated from experimental SAH and sham-operated rats. Following experimental SAH, cerebral arteries exhibited increased vasoconstriction to endothelin-1, 5-hydroxytryptamine and U46419. In addition, depolarization-induced vasoconstriction (60 mM potassium) was significantly increased, supporting a general SAH-associated vasoconstrictive phenotype. Using exogenous CGRP, we demonstrated that sensitivity of the arteries to CGRP-induced vasodilation was unchanged after SAH. However, vasodilation in response to capsaicin (100 nM), a sensory nerve activator used to release perivascular CGRP, was significantly reduced by SAH (P = 0.0079). Because CGRP-mediated dilation is an important counterbalance to increased arterial contractility, a reduction in CGRP release after SAH would exacerbate the vasospasms that occur after SAH. A similar finding was obtained with artery culture (24 h), an in vitro model of SAH-induced vascular dysfunction. The arterial segments maintained sensitivity to exogenous CGRP but showed reduced capsaicin-induced vasodilation. To test whether a metabolically stable CGRP analogue could be used to supplement the loss of perivascular CGRP release in SAH, SAX was systemically administered in our in vivo SAH model. SAX treatment, however, induced CGRP-desensitization and did not prevent the development of vasoconstriction in cerebral arteries after SAH.

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

The main purpose of this study was to compare in vitro pharmacological properties of human αCGRP (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 ±â€¯0.12 and 9.0 ±â€¯0.11, respectively). A corresponding difference in receptor binding affinity of SAX and CGRP was determined in rat cerebral membranes (pKi 8.3 ±â€¯0.19 and 9.3 ±â€¯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 ±â€¯0.18 and 9.5 ±â€¯0.13, respectively) and human recombinant receptors (cAMP signalling pEC50 9.1 ±â€¯0.16 and 10.2 ±â€¯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.

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.

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.

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

BACKGROUND: Upregulation of vasoconstrictor receptors in cerebral arteries, including endothelin B (ETB) and 5-hydroxytryptamine 1B (5-HT(1B)) 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. RESULTS: We 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. CONCLUSIONS: Our 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.