Jacob Hansen-Schwartz

Cerebral vasospasm following subarachnoid hemorrhage: time for a new world of thought

Abstract Objective: Delayed cerebral vasospasm has long been recognized as an important cause of poor outcome after an otherwise successful treatment of a ruptured intracranial aneurysm, but it remains a pathophysiological enigma despite intensive research for more than half a century. Method: Summarized in this review are highlights of research from North America, Europe and Asia reflecting recent advances in the understanding of delayed ischemic deficit. Result: It will focus on current accepted mechanisms and on new frontiers in vasospasm research. Conclusion: A key issue is the recognition of events other than arterial narrowing such as early brain injury and cortical spreading depression and of their contribution to overall mortality and morbidity.

Subarachnoid Hemorrhage Enhances Endothelin Receptor Expression and Function in Rat Cerebral Arteries.

OBJECTIVEInspired by organ culture-induced changes in the vascular endothelin (ET) receptor population, we investigated whether such changes occur in cerebral arteries in a rat subarachnoid hemorrhage (SAH) model. METHODSSAH was induced with injection of 250 &mgr;l of blood into the prechiasmatic cistern. After 2 days, the middle cerebral artery, basilar artery, and posterior communicating artery were harvested. Pharmacological studies were performed in vitro, and levels of messenger ribonucleic acid (mRNA) were quantified in real-time reverse transcriptase-polymerase chain reaction assays. RESULTSIn the middle cerebral artery and basilar artery from rats with induced SAH, enhanced biphasic responses to ET-1 were observed. The −log(50% effective concentration) value for the high-affinity phase was approximately 12, compared with approximately 8.5 for sham-operated animals. At a concentration of ET-1 of 10−9 mmol/L (approximately equal to the physiological concentration of ET-1 in the plasma), submaximal contractions of 50 to 75% of the contraction obtained through stimulation with 60 mmol/L K+ were now observed. Quantitative mRNA studies with the same arteries demonstrated significant increases in the number of copies of ETB receptor mRNA but not ETA receptor mRNA. Evidence of functional ETB receptors was provided in antagonist studies. The posterior communicating artery did not exhibit significant changes. CONCLUSIONThe altered receptor profile observed may represent the final stage in the series of events leading from SAH to actual spasm of the artery. The pharmacological data for the ETB receptor suggest complex interactions between normally present ETA receptors and up-regulated ETB receptors.

Individual changes in lamotrigine plasma concentrations during pregnancy

Eleven pregnant women on lamotrigine (LTG) monotherapy were retrospectively reviewed. A significant decrease in the ratio of plasma LTG concentration-to-dose by 65.1% was observed during the second trimester (TM2) (p=0.0058) and by 65.8% during TM3 (p=0.0045) compared to pre-pregnancy values. Five patients experienced seizure deterioration during pregnancy. The pharmacokinetic changes display marked inter-patient variation, which stresses the importance of evaluating each woman individually by closely monitoring LTG concentrations until term.

ERK1/2 inhibition attenuates cerebral blood flow reduction and abolishes ET(B) and 5-HT(1B) receptor upregulation after subarachnoid hemorrhage in rat.

Upregulation of endothelin B (ETB) and 5-hydroxytryptamine 1B (5-HT1B) receptors via transcription has been found after experimental subarachnoid hemorrhage (SAH), and this is associated with enhanced phosphorylation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK1/2). In the present study, we hypothesized that inhibition of ERK1/2 alters the ETB and 5-HT1B receptor upregulation and at the same time prevents the sustained cerebral blood flow (CBF) reduction associated with SAH. The ERK1/2 inhibitor SB386023-b was injected intracisternally in conjunction with and after the induced SAH in rats. At 2 days after the SAH, cerebral arteries were harvested for quantitative real-time polymerase chain reaction, immunohistochemistry and analysis of contractile responses to endothelin-1 (ET-1; ETA and ETB receptor agonist) and 5-carboxamidotryptamine (5-CT; 5-HT1 receptor agonist) in a sensitive myograph. To investigate if ERK1/2 inhibition had an influence on the local and global CBF after SAH, an autoradiographic technique was used. At 48 h after induced SAH, global and regional CBF were reduced by 50%. This reduction was prevented by treatment with SB386023-b. The ERK1/2 inhibition also decreased the maximum contraction elicited by application of ET-1 and 5-CT in cerebral arteries compared with SAH. In parallel, ERK1/2 inhibition downregulated ETB and 5-HT1B receptor messenger ribonucleic acid and protein levels compared with the SAH. Cerebral ischemia after SAH involves vasoconstriction and subsequent reduction in the CBF. The results suggest that ERK1/2 inhibition might be a potential treatment for the prevention of cerebral vasospasm and ischemia associated with SAH.

In depth pharmacological characterization of endothelin B receptors in the rat middle cerebral artery.

Whereas the endothelin A receptor is generally believed to mediate vasoconstriction; the endothelin B receptor seems elusive; both dilative and constrictive responses have been reported. Using the in vitro arteriograph, a method allowing compartmentalized study of vessel segments, segments of rat middle cerebral artery were cannulated with micropipettes, pressurized and luminally perfused. Vessel diameters were evaluated using a microscope equipped with an imaging system. Both intra- and extraluminal applications of endothelin-1 produced constriction. Intraluminal administration of a selective endothelin B receptor agonist sarafotoxin 6c in precontracted cerebral arteries and in the presence of the endothelin A receptor blocker FR139317 caused vasodilation in a concentration-dependent manner. Inhibition of nitric oxide synthase significantly reduced the dilation induced by sarafotoxin 6c, whereas inhibition of cyclooxygenase and endothelium-derived hyperpolarizing factor did not.

Protein kinase mediated upregulation of endothelin A, endothelin B and 5‐hydroxytryptamine 1B/1D receptors during organ culture in rat basilar artery

Organ culture has been shown to upregulate both endothelin (ET) and 5‐hydroxytryptamine 1B/1D (5‐HT1B/1D) receptors in rat cerebral arteries. The purpose of the present study was to investigate the involvement of protein kinases, especially protein kinases C (PKC) and A (PKA) in this process. The effect of inhibiting protein kinases during organ culture with staurosporine (unspecific protein kinase inhitor), RO 31‐7549 (specific inhibitor of classical PKCs) and H 89 (specific inhibitor of PKA) was examined using in vitro pharmacological examination of cultured vessel segments with ET‐1 (unspecific ETA and ETB agonist), S6c (specific ETB agonist) and 5‐CT (5‐HT1 agonist). Levels of mRNA coding for the ETA, ETB, 5‐HT1B and 5‐HT1D receptors were analysed using real‐time RT–PCR. Classical PKCs are critically involved in the appearance of the ETB receptor; co‐culture with RO 31‐7549 abolished the contractile response (6.9±1.8%) and reduced the ETB receptor mRNA by 44±4% as compared to the cultured control. Correlation between decreased ETB receptor mRNA and abolished contractile function indicates upstream involvement of PKC. Inhibition of PKA generally had an enhancing effect on the induced changes giving rise to a 7–25% increase in Emax in response to ET‐1, S6c and 5‐CT as compared to the cultured control. Staurosporine inhibited the culture induced upregulation of the response of both the ETA and the 5‐HT1B/1D receptors, but had no significant effect on the mRNA levels of these receptors. This lack of correlation indicates an additional downstream involvement of protein kinases.

Selective up-regulation of 5-HT(1B/1D) receptors during organ culture of cerebral arteries.

5-hydroxytryptamine (5-HT) is thought to be involved in migraine headache and the pathophysiology of cerebrovascular diseases. Previous data show that organ culture induces a phenotypic change in cerebral vessels. Therefore we investigated if these changes also applied for the vasoconstrictive 5-HT receptors. Rat cerebral arteries express 5-HT2 receptors. Using organ culture we observed a phenotypic change with a selective up-regulation of 5-HT1B/1D receptors. This was revealed by an increased sensitivity to the selective 5-HT1B/1D agonist 5-CT after organ culture (pEC50(fresh) 5.6 ± 0.2 and pEC50(cultured) 6.8 ± 0.4). The response was inhibited by the 5-HT1B/1D selective antagonist GR55562 (pEC50(fresh) 5.1 ± 0.2 and pEC50(cultured) 6.0 ± 0.3). The organ model might mimic the phenotypic changes during cerebrovascular diseases.

Protein kinase C inhibition prevents upregulation of vascular ET(B) and 5-HT(1B) receptors and reverses cerebral blood flow reduction after subarachnoid haemorrhage in rats.

The pathogenesis of cerebral ischaemia after subarachnoid haemorrhage (SAH) still remains elusive. The purpose of the present study was to examine whether specific protein kinas C (PKC) inhibition in rats could alter the transcriptional SAH induced Endothelin (ET) type B and 5-hydroxytryptamine type 1B (5-HT1B) receptor upregulation and prevent the associated cerebral blood flow (CBF) reduction. The PKC inhibitor RO-31-7549 or vehicle was injected intracisternally after the induced SAH in rats (n = 3 to 10 in each groups for each method). The involvement of the PKC isoforms was investigated with Western blot; only PKCδ and PKCα subtypes were increased after SAH RO-31-7549 treatment abolished this. At 2 days after the SAH basilar and middle cerebral arteries were harvested and the contractile response to endothelin-1 (ET-1; ETA and ETB receptor agonist) and 5-carboxamidotryptamine (5-CT; 5-HT1 receptor agonist) were investigated with a myograph. The contractile responses to ET-1 and 5-CT were increased (P < 0.05) after SAH compared with sham operated rats. In parallel, the ETB and 5-HT1B receptor mRNA and protein expression were significantly elevated after SAH, as analysed by quantitative real-time polymerase chain reaction and immunohistochemistry, respectively. Administration of RO-31-7549 prevented the upregulated contraction elicited by application of ET-1 and 5-CT in cerebral arteries and kept the ETB and 5-HT1B receptor mRNA and protein levels at pre-SAH levels. Regional and global CBF evaluated by an autoradiographic technique were reduced by 60%±4% after SAH (P < 0.05) and prevented by treatment with rO-31-7549. Our study suggests that PKC plays an important role in the pathogenesis of cerebral ischaemia after SAH.

Liraglutide, a long-acting GLP-1 mimetic, and its metabolite attenuate inflammation after intracerebral hemorrhage

The inflammatory response plays a pivotal role in propagating injury of intracerebral hemorrhage (ICH). Glucagon-like-peptide-1 (GLP-1) is a hormone with antidiabetic effect and may also have antiinflammatory properties. Despite consensus that the glucoregulatory action is mediated by the GLP-1 receptor (GLP-1R), mechanisms in the brain remain unclear. We investigated the effect of a long-acting GLP-1 analog, liraglutide, and its truncated metabolite, GLP-1(9–36)a from dipeptidyl peptidase-4 (DPP-4) cleavage in ICH-induced brain injury. Primary outcomes were cerebral edema formation, neurobehavior, and inflammatory parameters. GLP-1(9–36)a, GLP-1R inhibitor, adenosine monophosphate-activated protein kinase (AMPK) phosphorylation inhibitor and DPP-4 inhibitor were administered to examine the mechanisms of action. Liraglutide suppressed neuroinflammation, prevented brain edema and neurologic deficit following ICH, which were partially reversed by GLP-1R inhibitor and AMPK phosphorylation inhibitor. Liraglutide-mediated AMPK phosphorylation was unaffected by GLP-1R inhibitor, and was found to be induced by GLP-1(9–36)a. GLP-1(9–36)a showed salutary effects on primary outcomes that were reversed by AMPK phosphorylation inhibitor but not by GLP-1R inhibitor. Liraglutide and DPP-4 inhibitor co-administration reversed liraglutide-mediated AMPK phosphorylation and antiinflammatory effects. Liraglutide exerted duals actions and the antiinflammatory effects are partially mediated by its metabolite in a phosphorylated AMPK-dependent manner. Therapies that inhibit GLP-1 degradation may weaken the metabolite-mediated effects.

Increased sensitivity to ET-1 in rat cerebral arteries following organ culture.

Endothelin-1 (ET-1) is recognized as being involved in the pathophysiology of cerebrovascular diseases. Using organ culture as a model for possible pathological changes we studied changes in ETA and ETB receptor function using a sensitive in vitro method. We observed an up-regulation of the ETB receptor and an amazingly increased sensitivity to ET-1 by 3 log units in pEC50; pEC50(fresh) was 8.7 ± 0.1, and pEC50(cultured) was 11.7 ± 0.3. pA2 for FR139317 in the fresh vessel was 7.0 ± 0.2 whereas it could not be obtained for the cultured vessel, indicating a possible cross-talk between the ETA and ETB receptors. The increased sensitivity to ET-1 could also take place during cerebrovascular disease such as stroke or haemorrhage rendering the vessels considerably more sensitive to ET-1.