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Kevin S. Lee and Murad Bavbek

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Tomikatsu Toyoda, Neal F. Kassell, and Kevin S. Lee

Object. Inflammatory responses and oxygen free radicals have increasingly been implicated in the development of ischemic brain injury. In some cases, an attenuation of inflammation or free-radical injury can provide tissue protection. Diphosphoryl lipid A (DPL) is a detoxified derivative of a lipopolysaccharide (endotoxin) of Salmonella minnesota strain R595, which is capable of stimulating the immune system without eliciting direct toxic effects. In this study the authors examined the influence of preconditioning with DPL on ischemia/reperfusion injury in rats.

Methods. Sprague—Dawley rats were injected intravenously with either DPL or vehicle. Twenty-four hours later, some animals were tested for superoxide dismutase (SOD) activity. Others were subjected to a 3-hour period of focal cerebral ischemia and, after a reperfusion period of 24 hours, were killed. Infarction volume, SOD activity, and myeloperoxidase (MPO) activity were assayed in the postischemic animals.

Pretreatment with DPL produced significant reductions in cerebral infarction and MPO activity in the ischemic penumbra. A significant enhancement of basal SOD activity was observed 24 hours after DPL treatment (that is, before ischemia), and a further enhancement of SOD activity was seen in the ischemic penumbra 24 hours after reperfusion.

Conclusions. These data provide the first evidence of a neuroprotective effect of preconditioning with DPL in an in vivo model of cerebral ischemia. Although the precise mechanisms through which DPL exerts its neuroprotective influence remain to be established, an inhibition of the complex inflammatory response to ischemia and an enhancement of endogenous antioxidant activity are leading candidates.

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Satoshi Suzuki, Neal F. Kassell, and Kevin S. Lee

✓ Hemin is a prominent breakdown product of hemoglobin, and high levels of hemin are found in the cerebrospinal fluid during subarachnoid hemorrhage—induced vasospasm. The possible role of hemin in modifying vascular function was examined in the present study by testing its effects on nitric oxide synthase (NOS) activity in cultured rat aortic smooth-muscle cells. Nitric oxide synthase activity was estimated from the amounts of accumulated nitrite and nitrate, which are oxidative products of nitric oxide (NO). Hemin (1–100 µM) increased the levels of nitrite and nitrate in culture medium in a dose- and time-dependent manner. The hemin-induced elevation of nitrite and nitrate was inhibited significantly by the NOS inhibitor, Nω-nitro-l-arginine (300 µM), and by the protein synthesis inhibitor, cycloheximide (5 µg/ml). These results indicate that hemin is capable of stimulating the expression of an inducible isoform of NOS (iNOS) in vascular smooth muscle. Transcriptional expression of iNOS is known to cause injurious effects on the maintenance of cellular homeostasis by generating extremely high levels of NO. The generation of hemin from methemoglobin during hemolysis of a subarachnoid blood clot could therefore stimulate an excessive production of NO in vascular smooth-muscle cells. It is postulated that this series of events contributes to the development of vascular injury associated with cerebral vasospasm after aneurysmal subarachnoid hemorrhage.

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Bernhard Sutter, Satoshi Suzuki, Neal F. Kassell, and Kevin S. Lee

✓ Increasing evidence suggests that disturbances in the modulatory influence of the vasoactive peptide, calcitonin gene—related peptide (CGRP), contribute to the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH). However, only limited success has been achieved in trials attempting to ameliorate vasospasm by modifying CGRP function. To better understand the potential utility of targeting CGRP-mediated relaxation, it is important both to identify the interactions CGRP may have with other elements of the vasospastic response and to characterize the mechanisms through which CGRP elicits vasodilative effects. The present studies examined the effects of CGRP on vascular responsiveness using tension measurements of ring strips of rabbit basilar artery maintained in vitro. Pretreatment of vessels with CGRP (100 nM) inhibited vasoconstrictor responses to the potent protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDB). This particular contractile response was selected because PKC-mediated vasoconstriction is a critical component of the vasospastic response after SAH. In a posttreatment paradigm, CGRP was also found to reverse established constriction responses to PDB (2 nM) and histamine (3 µM) in a dose-dependent manner.

When tested against the maximum effective dose of PDB (30 nM) in the posttreatment paradigm, CGRP (100 nM) did not elicit significant relaxation. However, after washing both of these drugs out of the test chamber, a persistent effect of CGRP was revealed: the decay of PDB-induced contraction was accelerated in vessels that had previously been treated with CGRP. These findings indicate that CGRP elicits both immediate and sustained influences on contractile responses mediated by PKC.

Finally, two potential mechanisms for the vascular response to CGRP were examined. Adenosine triphosphate (ATP)—sensitive K+ channels do not appear to participate in CGRP-mediated dilation; inhibitors of these channels, glibenclamide and tolbutamide, did not block CGRP-induced relaxation. In contrast, a possible role for the nucleotide cyclic adenosine monophosphate (cAMP) in the vascular response to CGRP was indicated by the dose-dependent elevation of cAMP levels by CGRP.

Together these studies indicate that CGRP can modulate the contractile response to PKC activation. These effects are associated with increases in the levels of cAMP, but occur independently of fluxes through ATP-sensitive K+ channels.

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Satoshi Suzuki, Katsunobu Takenaka, Neal F. Kassell, and Kevin S. Lee

✓ The roles of hemoglobin (Hb) in the pathogenesis of cerebral vasospasm remain a matter of discussion. Hemoglobin is known to be released from extravasated red blood cells in a variety of pathological conditions, including subarachnoid hemorrhage. These conditions are often accompanied by infiltration of inflammatory cells and an associated release of multiple cytokines. Certain of these cytokines, including interleukin-1β (IL-1β), are capable of increasing nitric oxide (NO) production via the inducible form of nitric oxide synthase (NOS), and excessive NO production under these conditions may contribute to cellular dysfunction. This study further examines these questions by investigating the effects of Hb on the induction of NOS by IL-1β.

The effects of Hb on IL-1β-induced NO production were examined in cultured smooth-muscle cells of rat aorta (RA-SMC's). Production of NO was estimated from the accumulation of nitrite, an oxidative product of NO, in the culture medium. The synthesis of NO was induced by IL-1β in a concentration-dependent manner. This activation of NO production was inhibited by: 1) a general inhibitor of NOS (Nω-nitro-L-arginine); 2) a protein synthesis inhibitor (cycloheximide); and 3) two selective inhibitors of the inducible form of NOS (hydrocortisone and aminoguanidine). These results suggest that IL-1β promotes the expression of the inducible form of NOS in RA-SMC's. The effects of Hb on NO production were tested by adding purified human Hb to the culture medium of the cells in both the presence and absence of IL-1β. Nitrite accumulation was slightly but significantly increased by Hb in the absence of IL-1β. In contrast, Hb markedly augmented nitrite accumulation induced by IL-1β. This augmentation persisted even after the removal of Hb from the culture medium. The number of cells was not affected by Hb or IL-1β.

The findings demonstrate that Hb can modify cytokine-induced production of NO in RA-SMC's by increasing the inducible form of NOS. These observations suggest that Hb can also modify the action of inflammatory cells by facilitating NO production in target cells.

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Patricia L. Foley, Katsunobu Takenaka, Neal F. Kassell, and Kevin S. Lee

✓ The release of intracellular products from lysed blood cells is believed to play a critical role in the etiology of vascular pathology following intracerebral hemorrhage. The present studies investigated the effects of a mixture of blood and cerebrospinal fluid (CSF) on bovine intracranial endothelial cells maintained in culture. The incorporation of 3H-leucine into endothelial cells was used as an index of cellular viability. Cerebrospinal fluid alone did not alter the incorporation of 3H-leucine into the cells. In contrast, CSF preincubated with blood for 3 days or longer prior to treatment elicited significant reductions in leucine incorporation. Treatment with CSF preincubated with blood for 5 to 7 days resulted in the rapid deterioration of the culture, with large numbers of cells detaching almost immediately. Concentrations of hemoglobin were elevated profoundly in mixtures of blood and CSF preincubated for periods longer than 3 days. The increases in hemoglobin concentration were related temporally to increases in the cytotoxic impact of the bloody CSF.

These findings suggest that factors released during the breakdown of blood exert a deleterious effect on intracranial endothelial cells. The time course of this effect is closely related to the development of vasospasm in humans following subarachnoid hemorrhage. Taken together, these observations are consistent with the hypothesis that intracellular blood products, particularly hemoglobin, contribute to vasospasm by directly compromising endothelial function.

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Shinsaku Nishio, Masatoshi Yunoki, Zong-Fu Chen, Matthew J. Anzivino, and Kevin S. Lee

Object. Ischemic neuronal damage associated with neurological and other types of surgery can have severe consequences for functional recovery after surgery. Hypothermia administered during and/or after ischemia has proved to be clinically beneficial and its effects often rival or exceed those of other therapeutic strategies. In the present study the authors examined whether transient hypothermia is an effective preconditioning stimulus for inducing ischemic tolerance in the brain.

Methods. Adult rats were subjected to a 20-minute period of hypothermic preconditioning followed by an interval ranging from 6 hours to 7 days. At the end of this interval, the animals were subjected to transient focal ischemia induced by clamping one middle cerebral artery and both carotid arteries for 1 hour. The volume of cerebral infarction was assessed 1 or 7 days postischemia. In the first series of experiments, hypothermic preconditioning (28.5°C) with a postconditioning interval of 1 day reduced the extent of cerebral infarction measured 1 and 7 days postischemia. In the second series, hypothermic preconditioning (31.5°C) with postconditioning intervals of 6 hours, 1 day, or 2 days (but not 7 days) reduced the extent of cerebral infarction measured 1 day postischemia. Treatment with the protein synthesis inhibitor anisomycin blocked the protective effect of hypothermic preconditioning. In a final series of experiments, in vitro brain slices prepared from hypothermia-preconditioned (nonischemic) animals were shown to tolerate a hypoxic challenge better than slices prepared from unconditioned animals.

Conclusions. These findings indicate that hypothermic preconditioning induces a form of delayed tolerance to focal ischemic damage. The time course over which tolerance occurs and the ability of a protein synthesis inhibitor to block tolerance suggest that increased expression of one or more gene products is necessary to establish tissue tolerance following hypothermia. The attenuation of hypoxic injury in vitro following in vivo preconditioning indicates that tolerance is due, at least in part, to direct effects on the brain neuropil. Hypothermic preconditioning could provide a relatively low-risk approach for improving surgical outcome after invasive surgery, including high-risk neurological and cardiovascular procedures.

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Masatoshi Yunoki, Shinsaku Nishio, Naoya Ukita, Matthew J. Anzivino, and Kevin S. Lee

Object. A brief period of hypothermia has recently been shown to induce delayed tolerance to ischemic brain injury. This form of tolerance is initiated several hours after hypothermic preconditioning (HPC) and persists for a few days. Hypothermia-induced tolerance could provide a means for limiting cellular injury during predictable periods of ischemia, such as those that occur during many surgical procedures. The purpose of this study was to characterize the parameters of HPC that regulate the induction of delayed tolerance.

Methods. The general design of the experiments was to perform HPC or a sham procedure on adult Sprague-Dawley rats. Twenty-four hours later, the animals were subjected to a transient period of ischemia induced by a 1-hour period of three-vessel occlusion. Infarct volume was assessed 24 hours postischemia. In the first series of experiments, the depth of global (that is, whole-body) HPC was set at 25.5, 28.5, or 31.5°C, and the duration of HPC was fixed at 20 minutes. In the second series of experiments, the duration of global HPC was set at 20, 60, 120, or 180 minutes, and the depth of HPC was set at 33 or 34.5°C. In the third series of experiments, focal HPC was administered by selectively cooling the head to achieve a cortical temperature of 28.5 or 31.5°C for 20 minutes, with the duration of HPC fixed at 20 minutes.

The magnitude of tolerance induced by HPC was dependent on the depth and duration of the hypothermic stimulus. The parameters of hypothermia that are capable of inducing tolerance are similar to, or less severe than, those already in clinical use during intraoperative procedures. Focal cooling was as effective as global cooling for eliciting tolerance, indicating that it is possible to establish tolerance while limiting the potential complications of systemic hypothermia.

Conclusions. The results of these experiments indicate that HPC may provide an effective and safe means for limiting cellular injury resulting from predictable periods of central nervous system ischemia.

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Bernhard Sutter, Satoshi Suzuki, Adam S. Arthur, Neal F. Kassell, and Kevin S. Lee

✓ Calcitonin gene—related peptide (CGRP) is a potent vasodilator and a primary signaling molecule in neurovascular communication. In the present study, the authors examined cerebrovascular responses to CGRP and its related second messenger systems during cerebral vasospasm induced by subarachnoid hemorrhage (SAH). Tension measurements were performed in vitro on ring strips of basilar arteries obtained from rabbits subjected to artificial SAH and from control (non-SAH) animals. In vessels from SAH animals, which were preconstricted with serotonin, the vasorelaxant response to CGRP was attenuated. Because it has been suggested that vasodilation elicited by CGRP is mediated by cyclic 3′,5′-adenosine monophosphate (cAMP) and/or cyclic 3′,5′-guanosine monophosphate (cGMP), the vascular effects of directly activating these second messenger systems were also examined. The relaxant effect of forskolin, which activates adenylate cyclase directly, was slightly enhanced after SAH. In contrast, the relaxant effect of nitroglycerin (GTN), which activates soluble guanylate cyclase directly, was unchanged after SAH.

The attenuation of CGRP-induced vasorelaxation could be the result of a modification in its ability to stimulate the production of second messengers. Experiments testing the capacity of CGRP to elevate cAMP levels showed no significant differences between vessels from non-SAH and SAH animals. Similarly, the resting levels of cAMP and the forskolin-induced elevations of cAMP did not differ between non-SAH and SAH animals. In contrast, cGMP levels were lower in resting and CGRP-treated vessels from SAH animals than in those from non-SAH animals. No significant differences in the levels of cGMP were observed between non-SAH and SAH vessels treated with GTN.

This study indicates that CGRP-induced vasodilation is attenuated during vasospasm in a rabbit model of SAH. The findings also demonstrate that vasodilatory responses mediated by cAMP and cGMP are intact, although the levels of cGMP in SAH vessels are reduced. Together, these observations suggest that an attenuation in the capacity of vessels to dilate in response to CGRP occurs during cerebral vasospasm, and this change in CGRP vasoactivity is a result of modifications prior to, or independent of, the elevation of cyclic nucleotide second messengers.

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Richard S. Polin, Murad Bavbek, Mark E. Shaffrey, Kevin Billups, Christopher A. Bogaev, Neal F. Kassell, and Kevin S. Lee

Object. The goal of this study was to explore whether the levels of soluble adhesion molecules were elevated in cerebrospinal fluid (CSF) after subarachnoid hemorrhage (SAH). This association was suggested by the known inflammatory response in vasospasm and the role of vascular adhesion molecules in regulating leukocytic adhesion to, and migration across, vascular endothelium.

Methods. A prospective analysis was performed on CSF samples obtained in 17 patients who had suffered a recent aneurysmal SAH and in 16 control patients by using quantitative enzyme-linked immunosorbent assays for E-selectin, intercellular adhesion molecule—1 (ICAM—1), vascular adhesion molecule—1 (VCAM-1), and L-selectin.

Levels of soluble forms of E-selectin (p = 0.0013), ICAM-1 (p = 0.0001), and VCAM-1 (p = 0.048) were found to be elevated in the CSF of patients after SAH compared with levels in the CSF of normal controls, patients with unruptured aneurysms, and patients tested months after SAH occurred. In addition, individual patients tested at the time of their initial ictus demonstrated a fall in adhesion molecule levels over time. Levels of E-selectin (p = 0.044) were highest in patients who later developed moderate or severe vasospasm.

Conclusions. Adhesion molecules are known to be involved in white cell adherence to the endothelium and subsequent diapedesis and migration in which a role in initiation of tissue damage is postulated. The authors have demonstrated the elevation of three adhesion molecules, with severely elevated levels of E-selectin seen in patients who later develop vasospasm. A correlation with a role of vascular adhesion molecules in the pathogenesis of cerebral vasospasm is suggested.