Brain Poster Session: Subarachnoid Hemorrhage

25. The effect of energy metabolism and intracellular free magnesium of brain after good grade subarachnoid haemorrhage

G. Wong, D. Yeung, A. Ahuja, A. King, M. Chan, C. Lam and W. Poon

The Chinese University of Hong Kong, Hong Kong, Hong Kong S.A.R.

Objective: Disturbance in energy status happens in many disease entities and had not been previously investigated in patients with subarachnoid hemorrhage. Pilot studies had indicated that hypermagnesemic treatment may improve outcome in patients with aneurysmal subarachnoid hemorrhage but the exact mechanism is not known.

Methods: We designed the current studies to investigate the energy metabolism and intracellular free magnesium using ³¹P MRS Brain, in good grade aneurysmal subarachnoid hemorrhage patients with and without hypermagnesemic treatment.

Results: A total of 35 eligible patients and 23 healthy volunteers were recruited into the study. A total of 102 MRS studies were performed. Hypermagnesemic treatment after aneurysmal subarachnoid hemorrhage produced a small (15.6%) but significant elevation of intracellular free magnesium during the first week. Aneurysmal subarachnoid hemorrhage produced a depressed cerebral metabolism with higher ATP/Pi and a depressed membrane metabolism with higher PDE/total phosphates, which recovered with time and lasted beyond the initial two weeks.

Interpretation: The finding of elevated brain free intracellular magnesium with intravenous magnesium sulfate infusion is novel. It provides a possible mechanism for intravenous magnesium sulfate infusion to improve outcome in patients with aneurysmal subarachnoid hemorrhage. The finding of depressed cerebral and membrane metabolism provides insight into the metabolic effects of aneurysmal subarachnoid hemorrhage and future pathophysiological studies.

357. Subarachnoid hemorrhage induces autophagy

J.-Y. Lee^1,2, Z. Chen¹, S. Hu¹, O. Sagher¹, R.F. Keep¹, Y. Hua¹ and G. Xi¹

¹Department of Neurosurgery, University of Michigan, Ann Arbor, Michigan, USA; ²Department of Neurosurgery, University of Cologne, Cologne, Germany

Objectives: Aneurysmal subarachnoid hemorrhage (SAH) is a serious disease leading to high morbidity and mortality. The most death occurs within few days, and the exact mechanisms underlying brain injury during this early period following SAH are not fully identified. ¹ Apoptotic cell death plays an important role in early brain injury after SAH. Recently, activated autophagy has been demonstrated in neurons exposed to intracerebral hematoma, hypoxia-ischemia, and traumatic brain injury. However, the role of autophagy is controversial. This study employed a modified endovascular perforation rat model to investigate whether autophagy pathway is involved in the early brain injury following SAH.

Methods: Male Sprague-Dawley rats were used. SAH was induced using endovascular perforation technique described by Bederson et al ² with slight modifications consisting of ICP-guided vessel perforation and temporarily occlusion of the ipsilateral ICA for 2 mins. Sham-operated control rats underwent an identical procedure without vessel perforation (n = 10). The rats were killed at Day 1 and 3 following SAH induction (n = 10, each group). Microtubule-associated protein light chain-3 (LC3) and cathepsin-D were investigated by Western blot analysis and immunohistochemistry. Electron microscopy was performed to examine the ultrastructural changes of the neurons after SAH.

Results: LC3-II is one of the biochemical markers for autophagy. Although LC3-I could be detected in sham-operated control rats, the conversion of LC3-I to LC3-II was significantly increased in the ipsilateral frontobasal cortex at Day 1 and Day 3 (P<0.05) following SAH induction. Cathepsin-D is a lysosomal enzyme important for degradation of altered cellular materials, and a significant elevated cathepsin-D expression could be demonstrated at Day 1 (P<0.01) as well as on Day 3 (P<0.05) compared to sham-operated control animals. Immunohistochemical study with antibody against cathepsin-D showed numerous positive stained neurons in deep layer of the frontobasal cortex. Under electron microscopy, double-membraned vacuoles were markedly accumulated in neurons at Day 1 following SAH.

Conclusion: This present study showed that the autophagy pathway is significantly activated in the acute phase after SAH. Further studies are needed to clarify the role of autophagy in SAH.

359. Deferoxamine reduces early brain injury following subarachnoid hemorrhage

J.-Y. Lee^1,2, Y. He¹, O. Sagher¹, R.F. Keep¹, Y. Hua¹ and G. Xi¹

¹Department of Neurosurgery, University of Michigan, Ann Arbor, Michigan, USA; ²Department of Neurosurgery, University of Cologne, Cologne, Germany

Objectives: The treatment of subarachnoid hemorrhage (SAH) remains one of the major challenges. Despite advances in the diagnosis and treatment of aneurysms, the pathophysiological aspects following SAH are still poorly understood, and the therapeutic options in the acute phase are limited. Recent experimental studies in the setting of intracerebral hemorrhage in rats have demonstrated that deferoxamine, an iron chelator, reduced significantly brain edema and neuronal cell death leading to improved neurological outcome. ¹ In this study, the effect of deferoxamine on early brain injury following SAH was examined.

Methods: Male Sprague-Dawley rats were used. SAH was induced using endovascular perforation technique described by Bederson et al ² with slight modifications consisting of ICP-guided vessel perforation and temporary occlusion of the ipsilateral ICA for 2 mins. The rats were treated with either deferoxamine (100 mg/kg, i.p., given 2 h after hemorrhage followed by every 12 h) or vehicle (n = 12, each group). Sham-operated control rats underwent an identical procedure without vessel perforation (n = 12). The rats were killed at Day 1 following SAH induction for measurement of brain edema. To assess oxidative brain injury and neuronal cell death, heme oxygenase (HO)-1 expression was analyzed using Western blot analysis and Fluoro-Jade immunofluorescence staining was performed.

Results: Twenty-four hours following SAH induction, a significant increase in water content could be determined in ipsilateral cortex (P<0.05) accompanied by significant upregulation of HO-1 expression (P<0.05) in comparison with sham-operated control animals. Furthermore, diffusely distributed dying cells throughout the ipsilateral hemisphere could be observed at Day 1 following SAH induction. The treatment with deferoxamine resulted in significant decrease in brain edema (P<0.05) and significant reduction in HO-1 expression (P<0.05) as well as markedly less Fluoro-Jade positive labeled neurons.

Conclusion: Additionally to global ischemia, the excessive hemoglobin and iron overload play an important role in early brain injury following SAH. Acute treatment with deferoxamine ameliorates significantly hemoglobin-induced secondary brain lesions and therefore, could be a useful adjunct in the treatment of SAH.

414. Cerebral salt wasting following subarachnoid hemorrhage: a rat model

S. Schlaffer¹, H. Tam², N. Sharma², J. Verbalis², M. Buchfelder¹ and A. Kleindienst¹

¹Department of Neurosurgery, University Erlangen-Nuremberg, Erlangen, Germany; ²Department of Medicine, Georgetown University, Washington, District of Columbia, USA

Objective: Aneurysmal subarachnoid hemorrhage (SAH) is associated with a high mortality and morbidity. Beside vasospasm, one third of patients will develop severe hyponatriemia, which has also been referred to as cerebral salt wasting (CSW). Both syndromes have been connected to the hypothalamic neuropeptide arginine-vasopressin (AVP). Utilizing in vivo microdialysis an established experimental model of SAH, we were able to demonstrate a central AVP release, which may contribute to pathophysiological events following SAH. ¹ The present study is designed to examine CSW in experimental SAH.

Methods: In male Wistar rats (250 to 350 g), SAH was induced by injecting 150 μl of autologous blood into the great cistern (SAH n = 16). In a second group of rats, SAH was induced twice, 24 h apart (double SAH n = 12). Control rats underwent sham procedure (sham n = 8). The rats were housed in metabolic cages, and the daily sodium excretion as well as sodium, osmolarity and AVP in serum were measured for 10 days. Statistical analysis was performed using the Mann Whitney rank sum test (SPSS software).

Results: Following SAH, sodium excretion was significantly increased on day 1 (P = 0.002) and on day 4 (P = 0.026) as compared to control rats (n = 8). Serum sodium (141.5 mmol/l day 0, 142.0 mmol/l day 5) and serum osmolarity (303.5 mosmol/kg day 0, 307.2 mosmol/kg day 5) remained stable, while the initial increase of AVP normalized over the investigation period (46.99 pg/ml day 0, 8.14 pg/ml day 5). Following double SAH, rats suffered increased morbidity and lost significant weight, while natriuresis was not significantly altered.

Conclusions: The rat model of SAH by injecting blood into the great cistern is suitable for studying the pathophysiology of CSW. Increased natriuresis paralleled the time course of early and late vasospasm known in this model. Double SAH does not enhance natriuresis but resulted in increased morbidity and weight loss thereby interfering with the assessment of electrolyte and fluid balance.

422. Role of vasopressin V1a receptors for development of secondary brain damage after subarachnoid hemorrhage in rats

K. Hockel¹, K. Schöller^1,2, R. Trabold^1,2 and N. Plesnila^1,2,3

¹Laboratory of Experimental Neurosurgery, Institute for Surgical Research, University of Munich Medical Center—Grosshadern, Ludwig-Maximilians-University; ²Department of Neurosurgery, University of Munich Medical Center—Grosshadern, Ludwig-Maximilians-University, Munich, Germany; ³Department of Neurodegeneration, Royal College of Surgeons in Ireland, Dublin, Ireland

Objectives: Mechanisms of early brain damage after subarachnoid hemorrhage (SAH) are poorly understood and mortality remains high. Arginine vasopressin (AVP) and its V1a receptores are involved in the development of secondary brain damage after traumatic brain injury ¹ and focal cerebral ischemia. ² Thus, the aim of the present study was to investigate the role of AVP for brain edema formation, functional outcome and mortality following experimental SAH in rats.

Methods: Male Sprague-Dawley rats were anesthetized, intubated, and mechanically ventilated. SAH was induced using the filament perforation model. Mean arterial blood pressure (MABP), intracranial pressure (ICP), and regional cerebral blood flow (rCBF) were continiously recorded up to 60 mins after SAH. Seconds prior to the insult animals received either a V1a receptor antagonist (V1880–1 MG, 40 μg/kg bw i.v.) or vehicle (0.9% NaCl) for 3 h. Brain water content was assessed by the wet and dry method 24 h after SAH (n = 8 per group). Functional deficits and mortality were analyzed over 7 days (n = 10 per group). Plasma vasopressin concentration was determined at t = 5, t = 30 and t = 120 mins post SAH.

Results: Within 5 mins after SAH plasma vasopressin increased 35-fold as compared to baseline (P<0.05). Inhibition of V1a receptors prevented brain edema formation of the ipsilateral hemisphere (P<0.05), resulted in a better neurological function (P<0.05) and reduced 7-day mortality from 50% (vehicle group) to 20% (P<0.05).

Conclusions: The present study shows that AVP is involved in the development of secondary brain damage after SAH via V1a receptors. The early systemic application of a V1a receptor antagonist lead to significantly reduced mortality and improved neurologic function, most likely due to reduced brain edema formation. Therefore V1a receptor antagonists may be potent novel drugs for the reduction of secondary brain damage after subarachnoid hemorrhage.

474. Optimization of cerebral blood flow by intra-aortic balloon pump counterpulsation during management of neurogenic pulmonary oedema associated with subarachnoid haemorrhage

M. Hachemi¹, C. Di Roio¹, S. Tixier-Wulf¹, A. Terrier¹, E. Bonnefoy-Cudraz² and F. Dailler¹

¹Department of Neurocriticale Care; ²Department of Cardiac Intensive Care, Hospices Civiles de Lyon, Lyon, France

Introduction: Neurogenic pulmonary oedema (NPE) is a well recognised complication of subarachnoid haemorrhage (SAH) and severe traumatic brain injuries. The incidence of NPE was reported to be 6% in a series of 457 patients with SAH. ¹ NPE is characterized as an acute, protein-rich lung oedema occurring shortly after cerebral injury. NPE may compromise haemodynamics, decrease cerebral blood flow and increase cerebral vasospasm which increases the morbidity of SAH. Yet, the precise pathogenetic mechanisms remain unclear, but the role of a sympathetic storm may be the most important cause.

Patients and methods: We report two patients with neurogenic pulmonary oedema and cardiac failure after subarachnoid haemorrhage treated with intra-aortic balloon pump counterpulsation (IABP) therapy.

Results:

Case 1: A 51 year old man was admitted to the cardiac intensive care unit for cardiac arrest with pulmonary edema. Coronarography was normal. CT scan showed an extensive SAH with intraventricular haemorrhage and secondary hydrocephalus. Initial Glasgow coma scale was 3 (Hunt & Hess grade V). Cerebral angiography showed an aneurysm of the anterior communicant artery. Chest radiograph showed frank pulmonary oedema. Echocardiography showed acute severe left ventricular dysfunction (ejection fraction (EF) 25%) associated with left ventricular apical ballooning (takotsubo-like myocardial dysfunction). The PaO2/ FIO2 ratio was 75 on admission. Despite conventional treatment (mechanical ventilation, dobutamine) IABP was necessary to treat cardiac failure and pulmonary edema. Four hours after the insertion of IABP, PaO2/ FIO2 ratio was 315. Left ventricular EF reached 50% 12 h after insertion. The IABP was removed after 17 h. Despite embolization of the aneurysm and treatment of intracranial hypertension by decompressive craniectomy, the patient death occurred at day 8.

Case 2: A 46 year old woman was admitted in ICU for SAH (Hunt & Hess grade IV). Her medical past history included diabetes, hypertension, coronaropathy (EF 50%) and she was treated with clopidogrel and acetyl salicilyc acid. Ten hours after admission, she developed arterial hypotension and pulmonary edema (cardiogenic and neurogenic). PaO2/ FIO2 ratio was 150. At day two, despite conventional therapy (mechanical ventilation and high dose of dobutamine, norepinephrine, epinephrine), EF was less than 20%. IABP was inserted to optimize haemodynamic function. Three hours after the insertion of the IABP, infusion of dobutamine, norepinephrine, epinephrine was stopped. Then the IABP was removed after four days. The patient was extubated at day 13, and was discharged of the ICU at day 18 with EF 45% and a good cardiac and neurologic outcome.

Conclusions: These two cases illustrate the potential usefulness of the intra-aortic balloon pump counterpulsation as an adjunctive therapy in neurogenic pulmonary edema and cardiac failure after subarachnoid haemorrhage.

485. Effects of inhibition of the myosin-light-chain kinase on brain-edema formation after subarachnoid hemorrhage in mice

S. Feiler¹, C. Ricken², H. Luhmann³, K. Engelhard², C. Werner², C. Kuhlmann³, N. Plesnila^1,4 and S. Thal²

¹Department of Neurosurgery and Walter Brendel Center for Experimental Medicine, University of Munich Medical Center—Grosshadern, Ludwig-Maximilians-University, Munich; ²Department of Anesthesiology; ³Institute for Physiology und Pathophysiology, Johannes Gutenberg-University, Mainz, Germany; ⁴Royal College of Surgeons in Ireland, Dublin, Ireland

Objectives: The precise mechanisms leading to blood-brain-barrier (BBB) breakage and vasogenic brain edema formation after the subarachnoid hemorrhage (SAH) are still unknown. Recent studies suggest that the contractile apparatus of endothelial cells may contribute to the disruption of the BBB. In an in-vitro BBB model hypoxia activated the myosin light chain kinase (MLCK) of cerebral endothelial cells. This mediated a contraction and change in conformation of the endothelial cytoskeleton and resulted in increased permeability of the BBB. Increased phosphorylation of the myosin light chain (MLC) may therefore contribute to impaired integrity of the BBB² after cerebral insults. The aim of this study was therefore to investigate the role of MLCK on brain edema formation after experimental SAH.

Methods: Male C57/Bl6 mice (23 to 25 g body weight) were studied and cared for prior to and at all stages of the experiments in compliance with the institutional guidelines and regulations of the Animal Care Committee of the District Government of Rheinland-Pfalz. Mice were put under anesthesia with medetomidin, midozolam, fentanyl, intubated, and mechanically ventilated. After preparation of the left bifurcation of external and internal carotid artery, a blunted 5/0 monofilament suture was introduced into the ECA and was advanced into the ICA under control of cerebral blood flow and ICP until perforation of the circle of Willis. Afterwards all wounds were closed and anesthesia was terminated by antagonisation with flumacenil, naloxon and atipamezol. The mice were randomly assigned (n = 8 per experimental group) with allocation concealment to either the specific MLCK inhibitor ML-7 (1 mg/kg body weight) or vehicle (normal saline solution). Treatment was injected intraperitoneally 1 h before and 6 h after SAH induction. 24 h after SAH the brain edema, ICP and neurological outcome were measured. Data are presented as mean±s.d.

Results: 24 h after SAH brain water content was significantly reduced by 1.2% in comparison to the control group (81%±0.6% in the control group). The average ICP was 10±2 mm Hg in comparison to 18±2 mm Hg in the control group. A significantly improvement of neurological outcome was present in mice treated with ML-7 as compared to vehicle treatment (2±1 points vs. vehicle: 6.5±4).

Conclusions: In the present study we demonstrated, that inhibition of the myosin light chain kinase reduced brain edema formation, decreased ICP, and improved neurological outcome following SAH. In vitro data indicate that these effects are caused by reduced permeability and improved stability of the BBB. Accordingly, the activation of MLCs could be the underlying mechanism leading to the opening of the BBB after SAH. Therefore our study suggests that MLCs inhibitors may represent a novel class of drugs suitable for the treatment of post-hemorrhagic brain edema.

492. Inflammatory signaling via bradykinin receptors contributes to the development of secondary brain damage after subarachnoid hemorrhage in mice

K. Schöller^1,2, S. Feiler², S. Anetsberger² and N. Plesnila^1,2

¹Department of Neurosurgery, University of Munich Medical Center—Grosshadern, Ludwig-Maximilians-University; ²Insitute for Surgical Research, University of Munich Medical Center-Grosshadern, Ludwig-Maximilians-University, Munich, Germany

Objectives: The kallikrein-kinin system is involved in inflammatory signaling after traumatic brain injury and cerebral ischemia via bradykinin B₂ receptors. The current study was conducted to clarify the role of both bradykinin receptor subtypes (B₁ and B₂) for the development of secondary brain damage after experimental subarachnoid hemorrhage (SAH).

Methods: 6–8 week old C57/Bl6 mice (controls) and bradykinin B₁- and B₂-receptor knock-out (−/−) mice were anesthetized, intubated and mechanically ventilated. The SAH was induced by endovascular puncture. Intracranial pressure (ICP, parenchymal probe) and cerebral blood flow (CBF, Laser-Doppler flowmetry) were continuously measured from 15 mins before until 15 mins after SAH. 24 h after SAH ICP measurement was repeated and animals were subsequently sacrificed to quantify the brain water content. Mortality, neurological function and postoperative weight gain was quantified over a period of 7 days after SAH.

Results: ICP and CBF did not differ between groups in the first 15 mins after SAH. 24 h after SAH, however, ICP (P<0.05) and brain water content (P<0.05 B₂−/−) was reduced in bradykinin receptor knock-out animals compared to controls. 7-day mortality was 43%, 71% (P<0.05 vs. controls), and 14% (P<0.05 vs. controls) in control, B₁−/−, and B₂−/− mice, respectively. The neurological function was significantly better in B₂−/− mice (P<0.05) compared to control and B₁−/− mice on the first postoperative day. B₂−/− animals also exhibited a better weight gain on postoperative day 4 whereas the B₁−/− group showed a lesser weight gain from postoperative 3 through 7 compared to control animals (P<0.05).

Conclusions: An inflammatory response mediated by bradykinin B₂ receptors contributes to the development of secondary brain damage after SAH. Thus, pharmacological inhibition of B₂ receptors might be a future candidate for the treatment of post-hemorrhagic cerebral edema.

541. RAF inhibition prevents activation of pro-inflammatory mediators in cerebral arteries after subarachnoid hemorrhage

A. Maddahi, S. Ansar and L. Edvinsson

Clinical Science, Experimental Vascular Research, Lund University, Lund, Sweden

Objective: Late cerebral ischemia that is developed 4 to 15 days after subarachnoid hemorrhage (SAH) is a major cause of morbidity and mortality. ¹ Inflammatory mediators have been shown to be involved in the development of cerebral ischemia via activation of the mitogen-activated protein kinase (MAPK) pathway. ² In the present study, we hypothesize that treatment with a specific raf inhibitor will prevent the cerebral ischemia involving suppression of the inflammatory responses.

Methods: SAH was induced by injecting 250 μl autologous blood into the prechiasmatic cistern. Two groups of treated animals were intracisternally injected with a specific raf inhibitor (SB-386023-b) at 6 h or 12 h after the induced SAH. After 48 h the cerebral arteries were harvested and activation of the inflammatory and extracellular matrix-related genes (inducible nitric oxide synthase (iNOS), metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP1), interleukin 6 (IL-6) and interleukin-1beta (IL-1ß) and the extracellular signal regulated kinase 1/2 (ERK1/2) were investigated by immunofluorescence staining and real-time PCR. In addition, the CBF was examined by an auto radiographic technique.

Results: The level of MMP-9, TIMP1, iNOS, IL6 and IL-1ß proteins were upregulated in the wall of the basilar artery after SAH. The enhanced expression of MMP-9, TIMP1 and IL-1ß were located in the smooth muscle cells while iNOS and IL6 were located in both smooth muscle cells and endothelium as verified by colocalization studies. In addition, we demonstrated that SAH dramatically increases the phosphorylation level of ERK1/2 in the smooth muscle cells and reduces the cerebral blood flow (CBF) in the rat. In vivo treatment with a raf inhibitor given at 6 h but not after 12 h after SAH prevented the SAH induced upregulation of matrix metalloproteinase (MMPs), proinflammatory cytokines and ERK1/2 proteins and normalized CBF.

Conclusion: Treatment of signal-transduction at 6 h injection with a specific raf inhibitor prevents the enhanced expression of proinflammatory mediators and extracellular matrix-related genes seen after SAH. This suggests a novel approach towards treatment of inflammation associated with SAH.

557. Effect of clazosentan on microthrombosis after experimental subarachnoid hemorrhage

G. Chen, J. Ai and R.L. Macdonald

Neurosurgery, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada

Background: Although clinical studies suggest clazosentan, a synthetic endothelin A receptor (ET_A) antagonist, attenuates development of cerebral vasospasm after subarachnoid hemorrhage (SAH), its effect on the secondary pathophysiological changes after SAH has not yet been investigated. Accumulating evidence has demonstrated that SAH-induced microthrombosis plays a role in the pathogenesis of delayed cerebral ischemia after SAH. To date, however, few studies focused on treatment of microthrombosis after experimental SAH.

Purpose: The purpose of this study was to determine the impact of clazosentan on the microthrombi formation after SAH in rats.

Methods: Adult male Sprague Dawley rats were divided into four groups:

• (1)

  control saline injected group (n = 6);

• (2)

  SAH group (n = 6);

• (3)

  SAH+vehicle group (n = 8) and

• (4)

  SAH+clazosentan group (n = 8).

SAH was induced by injection of 0.3 ml fresh arterial, non-heparinized blood into the prechiasmatic cistern over 20 seconds with a syringe pump. In SAH+clazosentan group, clazosentan was administered as an intravenous bolus of 1 mg/kg 1 h after SAH and maintained at a constant infusion of 1 mg/kg/h with osmotic pump via the jugular catheter for 7 days. Brain samples were excised after perfusion fixation for further determinations at 7 days after SAH. Cross-sectional area of middle cerebral artery, basilar artery, and anterior cerebral artery was measured. Microthrombosis was evaluated by hematoxylin and eosin and fibrin (ogen) immunostaining.

Results: Clazosentan prevented vasospasm 7 after SAH (P<0.01). The number of microthrombi was significantly higher in both cerebrum and cerebellum 7 days after SAH in the SAH and control SAH+vehicle groups (P<0.01). Clazosentan decreased formation of microclots in this SAH model, and the number of microthombi-rich animals was decreased significantly in SAH+clazosentan group compared with SAH or SAH+vehicle groups (P<0.01).

Conclusion: Clazosentan attenuated cerebral vasospasm and alleviated the microthrombosis in the late phase of SAH in this prechiasmatic blood injection SAH model.

558. Alteration in voltage-dependent calcium channels in dog basilar artery after subarachnoid hemorrhage

R.L. Macdonald and E. Nikitina

Neurosurgery, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada

Background and aims: It is widely believed that nimodipine does not decrease vasospasm after subarachnoid hemorrhage (SAH), despite the prominent role of voltage-dependent calcium channels (VDCC), which it blocks, in contracting cerebral arteries. VDCC in large cerebral arteries have never been characterized, however, to determine why this is.

Methods: We characterized VDCC currents in smooth muscle cells from normal basilar artery and compared them to currents from cells obtained during vasospasm 7 days after SAH in dogs (n = 14 to 17 dogs per group). Whole cell currents were evoked by step-depolarizations from holding potential (V_h) of −50 or −90 mV in 10 mmol/L barium.

Results: Depolarization from V_h = −90 mV revealed currents with features of high (HVA, classically L-type) and low-voltage activated (LVA, classically T-type) VDCC with 25 of 147 cells having LVA current forming more than 50% of the maximal current and absent LVA current in another 25 cells. Other cells had mixtures of currents. Activation thresholds, deactivation kinetics and activation/inactivation taus also suggested 2 VDCC currents and demonstrated window current through LVA VDCC at physiological membrane potentials. In SAH cells, VDCC currents had lower peak amplitude than control cells and displayed dramatic reductions in voltage dependence of inactivation that caused corresponding reductions in calculated window current to less than 10% of control.

Conclusions: These novel data document the presence of LVA VDCC in basilar artery, which are typically less sensitive to nimodipine. Second, the marked reduction in window current suggests that even if calcium influx through VDCC initiates vasospasm, SAH may reduce VDCC current, rendering vasospasm less sensitive to VDCC antagonists.

655. Cerebral ischemia enhances vascular thromboxane receptor expressions

S. Ansar^1,2, C. Larsen² and L. Edvinsson^1,2

¹Clinical Sciences, Division of Experimental Vascular Research, Lund University, Lund, Sweden; ²Clinical Experimental Research, Glostrup Research Institute, Glostrup University Hospital, Glostrup, Denmark

Objectives: Cerebral ischemia remains the key cause of morbidity and mortality after subarachnoid hemorrhage (SAH) with a pathogenesis that is still poorly understood. Several studies have shown an increased thromboxane biosynthesis in patients with cerebral ischemia. We hypothesise that SAH induces changes in cerebrovascular receptor expression and function, which might influence the development of late cerebral ischemia. In previous studies have we revealed an upregulation of contractile endothelin type B (ET_B), angiotensin type 1 (AT₁) and 5-hydroxytryptamine 1B (5-HT_1B) receptors after SAH and temporal middle cerebral artery occlusion. The aim of the present study was to examine the involvement of thromboxane A₂ receptors (TP) in the pathofysiology of cerebral ischemia after SAH in cerebral arteries.

Methods: SAH was induced by injecting 250 μl blood into the prechiasmatic cistern. Two days after the SAH, basilar arteries (BA) and middle cerebral arteries (MCA) were harvested and contractile responses to the TP receptor agonist U46619 were investigated with myographs. In addition, the contractile responses were examined after pretreatment with the TP receptor antagonist GR3219b. The TP RNA levels were analyzed by quantitative real-time PCR. The global and regional cerebral blood flow (CBF) was quantified with an autoradiographic technique.

Results: SAH resulted in enhanced contractile responses to U46619 as compared to sham in BA and MCA. The TP receptor antagonist GR3219b abolished the enhanced contractile responses to U46619 observed after SAH. The TP receptor mRNA levels were elevated after SAH as compared to sham. Global and regional CBF was significantly reduced in SAH as compared to sham operated rats. Statistical analyses were performed with Kruskal-Wallis non-parametric test with Dunn's post-hoc test, where P<0.05 was considered significant. Data are expressed as mean±s.e.m.

Conclusion: The results demonstrate for the first time that SAH induces upregulation of TP receptors both at functional, and mRNA levels. These results suggest a role for the TP receptors in the pathogenesis of cerebral ischemia after SAH.

741. Detection and characterization of microvasospasm following subarachnoid hemorrhage in mice

B. Friedrich¹ and N. Plesnila^1,2

¹Institute for Surgical Research; ²Department of Neurosurgery, University of Munich Medical Center—Großhadern, Munich, Germany

Objective: Subarachnoid hemorrhage (SAH) has a mortality of over 60% within the first 48 after the initial bleeding. Mortality and morbidity are highly associated with a decrease of cerebral blood flow (CBF). However, since cerebral perfusion pressure (CPP) is often normal and vasospasm of the large vessels has not developed yet, the mechanisms of SAH-induced cerebral ischemia is still unclear. The hypothesis of the current study was that early post-hemorrhagic ischemia is mostly caused by constriction of the microvasculature, i.e. microvasospasm. Therefore the aim of this study was to develop an experimental model for the visualization of the cerebral microvasculature and to characterize microvascular changes after SAH.

Methods: SAH was induced in intubated and ventilated male C57/Bl6 mice by the previously described filament perforation technique. 3 h later the pial microcirculation was visualized through a cranial window using fluorescein isothiocyanate (FITC) dextran. A modified ‘Strahler-System’ was used to classify cerebral arterioles according to their diameter. Thereby the diameters of resistance vessel in sham-operated mice could be compared to those of mice following SAH.

Results: Already three hours after induction of SAH cerebral arterioles were significantly constricted as compared to control animals. Vessels of ‘Strahler’-category 3 (∼20 μm) showed a significant reduction of their diameter (21.86±0.77 mm vs. 18.27±1.13 mm; P<0.05). A total of 115 microvasospasm were detected out of 355 investigated resistance vessels. Microvasospasms were segmental, showed pearl-string like pattern, and were associated with paravascular blood. The smallest vessels (10 to 20 μm) showed the most significant microvasospasm (61.8%±2.4% vs. baseline; P<0.05) while the largest investigated vessels showed the weakest constriction (76.8%±1.9% vs. baseline; P<0.05).

Conclusion: We developed a model for the visualization of the cerebral microcirculation following SAH and detected already 3 h after SAH severe and significant changes of the cerebral microcirculation, i.e. microvasospasm of cerebral resistance vessels as previously detected in man (Uhl et al, Neurosurgery, 2003, 52(6)). We suggest that these microvasospasms are responsible for post-hemorrhagic ischemia and the related early mortality and morbidity of SAH. Further investigations using our novel model will need to identify the mechanisms of this phenomenon and show if post-hemorrhagic microvasospasm progresses towards vasospasm of larger cerebral vessels.

823. Cervical spinal cord stimulation for prevention of cerebral vasospasm in aneurysmal subarachnoid haemorrhage: preliminary results of first north american study

K. Slavin, E. Goellner, P. Eboli, S. Aydin, E. Colpan, N. Mlinarevich, K. Watson, R. Deveshwar, S. Amin-Hanjani and F. Charbel

Neurosurgery, University of Illinois at Chicago, Chicago, Illinois, USA

Objective: The goal of this study was to establish feasibility and safety of prolonged cervical spinal cord stimulation (SCS) in the setting of acute aneurysmal subarachnoid hemorrhage (aSAH), as well as to evaluate clinical effects of cervical SCS in a small group of selected aSAH patients. The study was undertaken in preparation for a larger scale randomized trial of SCS for prevention of cerebral arterial vasospasm following aSAH.

Material and methods: A single arm non-randomized prospective study of cervical SCS in aSAH patients was performed in University of Illinois at Chicago. Standard percutaneous 8-contact SCS electrodes were implanted under an Investigational Device Exemption protocol in 12 consecutive patients that satisfied the following inclusion criteria: age 18 to 65, angiography-confirmed aSAH within 3 days prior to the electrode implantation, Hunt/Hess (H&H) grade 2 to 4, Fischer grade 2 to 4, no history of previous cervical spine surgery, ability to obtain informed consent from the patient or family. All electrodes were inserted using percutaneous approach under general anesthesia immediately upon completion of the definitive surgical or endovascular procedure to secure the ruptured aneurysm. SCS was then delivered for the soonest of either 14 consecutive days or until the patient′s discharge. Daily vital signs, laboratory values, transcranial Doppler, computed tomography and angiography results were recorded along with the information on presence of clinical vasospasm and all interventions aimed at vasospasm prevention and treatment.

Results: Mean age of implanted patients was 49 years (range—27 to 62), average H&H grade—2.9, average Fisher grade—3.3. Three had aneurysms coiled and 9—clipped. One patient developed multisystem failure and expired on post-operative day 11. In two patients, electrode was inadvertently pulled out on days 7 and 13 after the implantation. There were no complications related to the electrode insertion or to SCS during the entire study period. The angiographic vasospasm was observed in 6 out of 12 patients, and clinical vasospasm—in 2 out of 12; no patient suffered any vasospasm-related neurological complication. Both incidences were smaller than predicted based on the patients′ Fisher and H&H grades.

Conclusions: This is the first North American study of SCS for prevention of vasospasm after aSAH; it conclusively shows both safety and feasibility of this promising invasive treatment approach. Our data indicate that despite high level of acuity in patients after aSAH, general severity of medical condition, impaired level of consciousness, frequent patient re-positioning, need in multiple tests and variety of monitors, SCS electrodes may be safely implanted and maintained for the two-week period. Further data analysis and additional studies will be needed to evaluate its therapeutic benefit.

908. High fat diet has little detrimental effect on stress gene expression post intracerebral hemorrhage

J. Clark¹, S. Benoit², A. Harm¹, D. Caudell¹, A. Lu¹ and G. Pyne-Geithman¹

¹Neurology; ²Psychiatry, University of Cincinnati, Cincinnati, Ohio, USA

We hypothesized that high fat diet would increase stress gene expression in the brains of rats post intracerebral hemorrhage (ICH). Obesity and diet are risk factors for having stroke and we wanted to determine whether high fat diet alone would have an impact on the recovery and/or complications seen post hemorrhage.

Using male rats 12 to 13 weeks of age we injected 50 ml of autologous arterial blood or an equal volume of normal saline for control. Normal chow consisted of 4.5% fat and high fat chow consisted of 20% fat. After the surgery the animals were allowed to recover. One group of animals on normal diet were sacrificed at 1 week post ICH and analyzed for hemoxygenase-1 (HO-1) and heatshock protein 25 (HSP-25). All other groups were aged for 6 weeks on either normal diet or high fat diet.

For all groups and all time points ICH induced significantly greater levels of HO-1 and HSP-25 compared to saline controls. When we compared high fat versus ICH there was no significant change in HO-1 expression and a slight not significant decrease in HSP-25 expression. In a post hoc analysis we found that the return towards saline control levels post ICH between 1 week and 6 weeks was similar for all groups.

In conclusion, we found that diet did not affect stress gene expression of HO-1 or HSP-25 following ICH.

932. Cerebral perfusion in patients after acute aneurysmal sah with or without intravenous magnesium sulfate infusion

G. Wong, R. Kwok, D. Yeung, A. King, A. Ahuja, K. Tang and W. Poon

The Chinese University of Hong Kong, Hong Kong, Hong Kong S.A.R.

Objective: A meta-analysis of current data suggests that magnesium sulfate infusion improves the outcome after aneurysmal subarachnoid hemorrhage through a reduction in delayed ischemic neurological deficit. Two multi-center randomized controlled trials are currently underway to investigate this hypothesis. The possible pharmacological basis of this hypothesis includes neuroprotection and vasodilatation. We aim to investigate the cerebral hemodynamic effects of magnesium sulfate infusion in aneurysmal subarachnoid hemorrhage patients.

Method: A total of 12 patients who had experienced aneurysmal subarachnoid hemorrhage were randomized to magnesium sulfate infusion (n = 6) or placebo infusion (n = 6) for 14 days. Each patient had two perfusion MRIs performed, one in the first week after subarachnoid hemorrhage and one in the second week after subarachnoid hemorrhage.

Results: Age, sex, and Fisher CT grade were not different between the two groups. All but one patient were of WFNS Grade I to II on presentation. No patient developed delayed ischemic neurological deficit. There was no increase in rCBV, rCBF and MTT between the two perfusion scans within the same group or between the two groups.

Conclusion: Magnesium sulfate infusion, in the dosage of current clinical trials, did not increase cerebral blood volume and cerebral blood flow, as postulated by dilation of small vessels and/or collateral pathways.

965. MRI of cerebral ischemia and chronic hyperperfusion after subarachnoid hemorrhage in rats

I.A.C.W. Tiebosch¹, W.M. van den Bergh², R. Zwartbol¹, K. Van Der Marel¹ and R.M. Dijkhuizen¹

¹Image Sciences Institute; ²Department of Intensive Care, University Medical Center Utrecht, Utrecht, The Netherlands

Objectives: Subarachnoid hemorrhage (SAH) carries poor prognosis, which has been attributed to vasospasm-related delayed cerebral ischemia. With MRI we have demonstrated that vasoconstriction, loss of cerebral blood flow (CBF) and ischemic tissue damage develop within 48 h after SAH in rats. ¹ Goal of the current study was to characterize the progression of tissue and perfusion changes in relation to outcome at chronic time-points after experimental SAH.

Methods: SAH was induced in isoflurane-anesthetized adult male Wistar rats (n = 17) by puncturing the right middle cerebral artery or the internal carotid artery at the level of their bifurcation, with a sharpened 4–0 polypropylene suture. ² MRI was conducted after 48 h and 7 days, and included multislice T₂- and diffusion-weighted MRI, dynamic susceptibility contrast-enhanced MRI in combination with an intravenous injection of gadobutrol, and pre- and post-contrast T₁-weighted MRI. Maps of the T₂, apparent diffusion coefficient (ADC), cerebral blood volume (CBV), CBF index (CBF_i) and contrast-induced T₁-weighted signal change were calculated ³ and co-registered to a stereotaxic rat brain atlas ⁴ . MR parameters were measured in ipsi- and contralateral primary somatosensory fore- and hindlimb regions (S1) and caudate putamen (CPu) and statistically analyzed with a paired Student's t-test.

Results: Ten rats died before the first MRI scan at 48 h, and four animals died between the first and second MRI scan at 7 days. At 48 h SAH-induced brain lesions were characterized by areas of reduced ADC and, more particularly, prolonged T₂, in large part of the ipsilateral somatosensory cortex and CPu (n = 7). Relative T₂ in ipsilateral S1 and CPu were 142%±26% and 132%±31%, respectively (% of contralateral; P<0.05). CBV and CBF_i were increased in these regions; on average by more than 40% (P<0.05). Also, there was significant contrast leakage in ipsilateral S1 and CPu. Three animals with significantly smaller ADC and T₂ alterations in the lesioned area as compared to the other four animals, survived between 2 and 7 days. In these animals, lesion T₂ and ADC values had largely normalized, but CBV and CBF_i remained elevated after 7 days.

Conclusions: Experimental SAH was accompanied by a relatively high mortality rate. In animals that survived until at least 48 h post-SAH, ADC reduction and T₂ prolongation in the MCA territory point toward cerebral ischemia. However, elevated CBV and CBF_i suggest that perfusion loss occurs before this time-point. Animals with least signs of ischemia survived beyond 48 h. Our study corroborates the significant role of cerebral ischemia in outcome after SAH. Hyperperfusion at later time-points, which has also been observed in aneurysmal SAH patients ⁵ , may be related to impaired autoregulation, but its effect on brain tissue status needs to be further investigated.

Funding:

Netherlands Heart Foundation (2005B156).

Netherlands Brain Foundation (15F07(2).08).

1041. Correlation between rCBF reduction obtained by 3D-SSP and neuropsychological impairment on the patients of subarachnoid hemorrhage treated by coil embolization

H. Yatsushige, Y. Takasato, H. Masaoka, T. Hayakawa, N. Otani, Y. Yoshino, T. Momose, C. Aoyagi and G. Suzuki

Neurosurgery, National Hospital Organization Disaster Medical Center, Tokyo, Japan

Objectives: Patients treated ruptured intracranial aneurysms often suffer from neuropsychological deficits in spite of a good neurological outcome. This study focuses on relates the neuropsychological test results to regional cerebral blood flow (rCBF) on 3D stereotactic surface projections (3D-SSP).

Methods: N-isopropyl-p-(123) I iodoamphetamine single photon emission computed tomography (IMP-SPECT) and neuropsychological examinations was performed in 16 patients with subarachnoid hemorrhage (SAH) treated by coil embolization (average age: 50 years old and average post-SAH days: 51). Z-score maps of IMP-SPECT images of a patient were obtained by comparison with data obtained from control subjects. The neuropsychological tests included the Wechsler Adult Intelligence Scale-Revised (WAIS-R) and Wisconsin Card Sorting Test (Keio version: KWCST).

Results: The mean of full scale Intelligence Quotient (IQ), performance IQ (PIQ), and verbal IQ (VIQ) of WAIS-R were 97.8, 90.7, and 96.9, respectively. Number of Categories Achieved (CA) of KWCST was poor in the 66% of patients. On statistical Z-score maps, the half of patients was significantly lower rCBF in cingulate gyrus, orbital gyrus, rectal gyrus, and uncus. A correlation was observed between the VIQ score and rCBF in left superior frontal gyrus and left medial frontal gyrus. The PIQ score was correlated with rCBF in right middle frontal gyrus. No correlation was showed between CA of KWCST and rCBF.

Conclusions: Previous study showed that surgical intervention significantly decreased mean rCBF in the operated frontal lobe compared with the unoperated side. However, even if all patients were treated with coil intervention in this study, rCBF in particular area was significant lower than control subjects. Therefore, these findings indicated that primary damage of SAH may result in the decrease of rCBF. Moreover, rCBF on 3D-SSP might be useful for prognosis of the neuropsychological impairment.