This is the first study to demonstrate that the duration of the initial CBF drop induced by injection of a standardised volume of blood into the prechiasmatic cistern is a determinant for a) the degree of ERK1/2 activation in cerebral arteries early after the SAH, b) delayed upregulation of vasoconstrictor receptors in cerebral arteries several days after the SAH and c) delayed CBF reduction, neurological deficits and mortality. Moreover, we show for the first time that treatment with an inhibitor of MEK-ERK1/2 signalling only during an early time window from 6 to 24 h after the SAH is sufficient to completely prevent delayed vasoconstrictor receptor upregulation and improve neurological outcome several days after the SAH. These findings suggest a series of events where 1) the drop in blood flow and wall tension experienced by the cerebral arteries during SAH triggers early activation of the MEK-ERK1/2 pathway, which 2) triggers increased expression and contractile function of vasoconstrictor receptors in cerebral arteries during the following days, where 3) the resulting enhanced cerebrovascular contractility contribute to development of delayed cerebral ischemia evident as CBF reduction, neurological deficits and mortality.
In this study, we investigate the different series of events taking place in two different ‘variants’ of the prechiasmatic injection SAH model differing in the duration of the acute CBF drop which was either short or prolonged. The occurrence of a prolonged acute CBF drop persisting after decline of the initial ICP rise is in accordance with earlier studies showing that acute vasoconstriction takes place after SAH. This can prolong the period of acute CBF reduction beyond the short time interval where ICP is increased to levels above jugular vein pressure [12, 13, 30, 31], a phenomenon that is also thought to take place in clinical acute SAH, at least in some patients. Since other important factors such as the amount of blood injected and the magnitude and duration of the initial ICP increase are kept constant, our experimental set-up reveals pathophysiological events triggered primarily by the acute CBF drop in itself, irrespective of hemorrhage volume and initial ICP increase.
We here demonstrate for the first time in this SAH modality that the duration of the initial CBF drop is a physiological determinant of neurological outcome and mortality during the first 4 days after SAH, a finding which is well in accordance with earlier studies using the endovascular perforation SAH model [12, 13]. However, this does not mean that the acute CBF drop is the sole determinant of delayed CBF reduction and delayed cerebral ischemia, and it is important to note that a number of studies have suggested that the amount of blood in the subarachnoid space and the rate of clearance of the blood clot determine the later risk of delayed cerebral ischemia and symptomatic CVS [8, 9], and thus the risk of delayed cerebral ischemia appears to be determined by a combination of multiple factors including, but not limited to, the duration of the acute CBF drop.
SAH induced both enhanced contractile function and increased protein expression of ETB and 5-HT1B receptors. We have earlier demonstrated that the increased receptor protein levels are associated with increased receptor mRNA levels [15, 16, 27], suggesting a transcriptional mechanism of upregulation, however, it cannot be ruled out that other mechanisms, such as reduced mRNA degradation, increased translation efficiency, and decreased receptor turnover, also play a role. We also show for the first time that the degree of cerebrovascular upregulation of ETB and 5-HT1B receptors during the first 3 days post-SAH depends strongly on the duration of the acute CBF drop. This suggests that the lack of flow and wall tension experienced by the cerebral arteries during the initial CBF drop may be the trigger of the receptor upregulation, rather than the exposure to extravascular blood in itself. This conclusion is in accordance with a recent study demonstrating that the degree of upregulation of cerebrovascular ETB and 5-HT1B receptors after transient occlusion of the two common carotid arteries combined with systemic hypotension is strongly dependent on the duration of the carotid artery occlusion . In support for a central role of the drop in vascular wall tension in the initiation of vascular ERK1/2 activation, we have recently shown that in a model of distal MCA occlusion contractile ETB receptors were upregulated only downstream from the occlusion, whereas the immediate upstream MCA, experiencing the same low degree of ischemia in the surrounding tissue but no drop in vascular wall tension, did not show changes in ETB receptor function. Moreover, we have recently demonstrated that the upregulation of contractile ETB receptors taking place during organ culture of cerebral artery segments can be prevented by applying a physiological level of wall tension to the artery segments during organ culture, and that this tension-dependent ETB upregulation is mediated by signalling via the focal adhesion kinase (FAK) known to be associated with integrin mechanosensitive protein complexes at the plasma membrane . These findings point to a vasogenic mechanosensitive trigger of ERK1/2 activation upon drop in wall tension in cerebral arteries, however, it cannot be ruled out that the decreased perfusion could induce the release of an endothelial factor, parenchymal metabolite, glial factor or neurohormone that act on the cerebral arteries to promote increased ERK activation.
The MEK-ERK1/2 signalling pathway has earlier been demonstrated to be involved in the upregulation of cerebrovascular ETB and 5-HT1B receptors after SAH. Thus, inhibition of either MEK1/2 or its upstream activator Raf completely prevents SAH-induced ERK1/2 activation and vasoconstrictor receptor upregulation in cerebral arteries and alleviates delayed cerebral ischemia [17, 18, 26]. The time-course of ERK1/2 activation in cerebral arteries after SAH was studied in detail in an earlier study, where increased ERK1/2 activity was demonstrated in cerebral arteries at time-points between 1-48 h post-SAH . However, the critical time window during which activation of this pathway drives the upregulation of vasoconstrictor receptors has not hitherto been investigated. Moreover, it has not been investigated whether the activation of the MEK-ERK1/2 pathway in cerebral arteries depends on the duration of the acute CBF drop during SAH. We here demonstrate activation of ERK1/2 in cerebral arteries throughout the first 6h post-SAH only in rats with prolonged acute CBF drops. Moreover, we show that treatment with a MEK1/2 inhibitor from 6 h to 24 h after SAH followed by a two days period without further treatment completely prevents the later enhancement of ETB- and 5-HT1B-mediated vasoconstriction in cerebral arteries. These findings, together with our demonstration of the importance of the acute CBF drop duration, suggest that the acute CBF drop induces early activation of the MEK-ERK1/2 pathway in cerebral arteries, which then during the time window from 6 to 24 h post-SAH acts as a ‘switch-on’ mechanisms for the expressional and functional upregulation of vasoconstrictor receptors in cerebral arteries over the following couple of days.
A large research effort has been put into findings effective treatments for CVS and delayed cerebral ischemia after SAH. Recently, the CONSCIUOS trials with the ETA receptor antagonist Clazosentan showed that specific targeting of ETA receptors is not sufficient to significantly alleviate delayed cerebral ischemia and improve clinical outcome after SAH [34, 35]. One possible explanation for the disappointing clinical effects of ETA receptor inhibition is that the complex vascular pathology after SAH involves many other, perhaps more or equally important, factors such as increased expression of several other vasoconstrictor receptors and their agonists , vascular inflammation , endothelial apoptosis and blood-brain barrier breakdown [37, 38]. The results of the present study underscore the importance of the acute phase of the SAH. We suggest that therapies targeting specific intracellular signal transduction components activated early after the SAH may help prevent the later evolution of SAH-induced vascular pathology contributing to delayed cerebral ischemia. Inhibition of the MEK-ERK1/2 pathway has in other studies been shown to alleviate delayed vascular inflammation, CBF reduction, and neurological deficits after experimental SAH [17, 18, 20]. The profound effect of MEK1/2 inhibition on vasoconstrictor receptor levels and neurological outcome when administered only from 6 to 24 h post-SAH in the present study, points to this as a possible way of targeting early changes within a clinically realistic therapeutic time window.