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  • Author or Editor: Aij-Lie Kwan x
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Tomikatsu Toyoda, Aij-Lie Kwan, Murad Bavbek, Neal F. Kassell, John E. Wanebo and Kevin S. Lee

Object. Monophosphoryl lipid A (MPL) and diphosphoryl lipid (DPL) are derivatives of the lipopolysaccharide (endotoxin) of Salmonella minnesota strain R595. Monophosphoryl lipid A is relatively nontoxic and can stimulate the natural defense or immune system. Diphosphoryl lipid is relatively toxic; however, at higher concentrations, it can also stimulate an immune response. The purpose of the present study was to determine the effects of these endotoxin analogs on cerebral vasospasm after the onset of subarachnoid hemorrhage (SAH) in rabbits.

Methods. Intrathecal administration of MPL or DPL (5 µg/kg) was performed immediately before and 24 hours after induction of SAH in New Zealand White rabbits. Forty-eight hours after induction of SAH, the animals were killed by perfusion fixation for morphometric analyses of vessels or perfused with saline and assayed for superoxide dismutase (SOD) activity. Additional rabbits were administered MPL or DPL and killed 24 hours later for assessment of SOD activity; no SAH was induced in these animals.

Experimental SAH elicited spasm of the basilar arteries in each group. Vasospasm was markedly attenuated in animals treated with MPL (p < 0.01 compared with vehicle-treated animals), but not in animals treated with DPL. A substantial reduction in SOD activity in the basilar artery accompanied the vasospasm; this loss of activity was significantly blocked by treatment with MPL, but not DPL. In animals that were not subjected to experimental SAH, MPL elicited a significant increase in SOD activity over basal levels, whereas DPL was ineffective.

Conclusions. These data provide evidence of a marked protective effect of the endotoxin analog MPL against vasospasm. Although the mechanism(s) responsible for the protective effect of MPL remains to be verified, an enhancement of basal antioxidant activity and an inhibition of SAH-induced loss of this activity are attractive candidates. An MPL-based therapy could represent a useful addition to current therapies for SAH-induced cerebral injury.

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Hakan H. Caner, Aij-Lie Kwan, Adam Arthur, Arco Y. Jeng, Rodney W. Lappe, Neal F. Kassell and Kevin S. Lee

✓ The potent vasoconstrictor peptide, endothelin-1 (ET-1), has been implicated in the pathophysiology of cerebral vasospasm that occurs after subarachnoid hemorrhage (SAH). This peptide is synthesized as a large prepropeptide that requires a series of modifying steps for its activation. The last of these steps involves the proteolytic conversion of a relatively inactive propeptide, Big ET-1, to its active, 21—amino acid peptide form. The enzyme responsible for converting Big ET-1 to ET-1 is a metalloprotease called endothelin-converting enzyme (ECE). In the present study the authors examined the effects of a newly developed inhibitor of ECE on responses to ET peptides in the normal basilar artery and on pathophysiological constriction in the spastic basilar artery after SAH.

In the first series of experiments the authors examined normal basilar arteries in the rabbit, which were exposed transclivally and measured on-line using videomicroscopy. Intravenous administration or topical application of an active inhibitor of ECE, CGS 26303, blocked vasoconstrictor responses to topically applied Big ET-1 but not to ET-1. In contrast, topical application of a structurally related compound that does not inhibit ECE, CGS 24592, was ineffective in blocking vasoconstriction that was elicited by a topical application of Big ET-1. These findings indicate that CGS 26303 when administered systemically is capable of blocking the conversion of Big ET-1 to ET-1 in the basilar artery without affecting the ability of the vessel to respond to ET-1. In the second series of experiments the authors examined the effects of the ECE inhibitor on cerebral vasospasm after experimental SAH. Intraperitoneal administration of CGS 26303 via osmotic minipumps significantly attenuated the delayed spastic response of the basilar artery to an intracisternal injection of autologous blood.

This study provides the first evidence that systemic administration of an inhibitor of ECE is capable of preventing cerebral vasospasm after SAH. The results reinforce a growing body of evidence that ETs play a critical role in the development of spastic constriction after SAH. Moreover, the findings indicate that blocking the conversion of Big ET-1 to its active ET-1 form using CGS 26303 may represent a feasible strategy for ameliorating cerebral vasospasm.

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Chih-Lung Lin, Aaron S. Dumont, Ann-Shung Lieu, Chen-Po Yen, Shiuh-Lin Hwang, Aij-Lie Kwan, Neal F. Kassell and Shen-Long Howng

Object. The reported incidence, timing, and predictive factors of perioperative seizures and epilepsy after subarachnoid hemorrhage (SAH) have differed considerably because of a lack of uniform definitions and variable follow-up periods. In this study the authors evaluate the incidence, temporal course, and predictive factors of perioperative seizures and epilepsy during long-term follow up of patients with SAH who underwent surgical treatment.

Methods. Two hundred seventeen patients who survived more than 2 years after surgery for ruptured intracranial aneurysms were enrolled and retrospectively studied. Episodes were categorized into onset seizures (≤ 12 hours of initial hemorrhage), preoperative seizures, postoperative seizures, and late epilepsy, according to their timing.

The mean follow-up time was 78.7 months (range 24–157 months). Forty-six patients (21.2%) had at least one seizure post-SAH. Seventeen patients (7.8%) had onset seizures, five (2.3%) had preoperative seizures, four (1.8%) had postoperative seizures, 21 (9.7%) had at least one seizure episode after the 1st week postoperatively, and late epilepsy developed in 15 (6.9%). One (3.8%) of 26 patients with perioperative seizures (onset, preoperative, or postoperative seizure) had late epilepsy at follow up. The mean latency between the operation and the onset of late epilepsy was 8.3 months (range 0.3–19 months). Younger age (< 40 years old), loss of consciousness of more than 1 hour at ictus, and Fisher Grade 3 or greater on computerized tomography scans proved to be significantly related to onset seizures. Onset seizure was also a significant predictor of persistent neurological deficits (Glasgow Outcome Scale Scores 2–4) at follow up. Factors associated with the development of late epilepsy were loss of consciousness of more than 1 hour at ictus and persistent postoperative neurological deficit.

Conclusions. Although up to one fifth of patients experienced seizure(s) after SAH, more than half had seizure(s) during the perioperative period. The frequency of late epilepsy in patients with perioperative seizures (7.8%) was not significantly higher than those without such seizures (6.8%). Perioperative seizures did not recur frequently and were not a significant predictor for late epilepsy.

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Chih-Lung Lin, Huei-Chuan Shih, Ann-Shung Lieu, Kung-Shing Lee, Aaron S. Dumont, Neal F. Kassell, Shen-Long Howng and Aij-Lie Kwan


Impaired endothelium-dependent relaxation is present in vasospastic cerebral vessels after subarachnoid hemorrhage (SAH) and may result from deficient production of endothelial nitric oxide synthase (eNOS) or increased production and/or activity of inducible NOS (iNOS). Accumulating evidence demonstrates that adenosine A2A receptors increase the production of NO by human and porcine arterial endothelial cells, which in turn leads to vasodilation. This study was designed to examine the effects of an adenosine A2A receptor agonist, (2(4-[2-carboxyethyl]phenyl)ethylamino)-5′-N-ethylcarboxamidoadenosine (CGS 21680), in the prevention of SAH-induced vasospasm.


Experimental SAH was induced in Sprague–Dawley rats by injecting 0.3 ml of autologous blood into the cisterna magna of each animal. Intraperitoneal injections of CGS 21680 or vehicle were administered 5 minutes and 24 hours after induction of SAH. The degree of vasospasm was determined by averaging measurements of cross-sectional areas of the basilar artery (BA) 48 hours after SAH. Expression of eNOS and iNOS in the BA was also evaluated.

Prior to perfusion–fixation, there were no significant differences among animals in the control and treated groups in any physiological parameter that was recorded. The CGS 21680 treatment significantly attenuated SAH-induced vasospasm. Induction of iNOS mRNA and protein in the BA by the SAH was significantly diminished by administration of CGS 21680. The SAH-induced suppression of eNOS mRNA and protein was also relieved by the CGS 21680 treatment.


This is the first evidence that adenosine A2A receptor agonism is effective in preventing SAH-induced vasospasm without significant complications. The beneficial effect of adenosine A2A receptor agonists may be, at least in part, related to the prevention of augmented expression of iNOS and the preservation of normal eNOS expression following SAH. Adenosine A2A receptor agonism holds promise in the treatment of cerebral vasospasm following SAH and merits further investigation.

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Aij-Lie Kwan, Murad Bavbek, Arco Y. Jeng, Wieslawa Maniara, Tomikatsu Toyoda, Rodney W. Lappe, Neal F. Kassell and Kevin S. Lee

✓ Delayed cerebral ischemia due to cerebral vasospasm is a major cause of morbidity and mortality in patients with aneurysmal subarachnoid hemorrhage (SAH). Increasing evidence implicates the potent vasoconstrictor peptide endothelin (ET) in the pathophysiology of cerebral vasospasm. In the present study the authors examined the therapeutic value of blocking the production of ET-1 by inhibiting the conversion of its relatively inactive precursor, Big ET-1, to a physiologically active form. An inhibitor of ET-converting enzyme (ECE), CGS 26303, was injected intravenously after inducing SAH in New Zealand white rabbits. Injections of CGS 26303 were initiated either 1 hour after SAH (prevention protocol) or 24 hours after SAH (reversal protocol). One of three concentrations (3, 10, or 30 mg/kg) of CGS 26303 was injected twice daily, and all animals were killed by perfusion fixation 48 hours after SAH occurred. Basilar arteries were removed and sectioned, and their cross-sectional areas were measured in a blind manner by using computer-assisted videomicroscopy.

Treatment with CGS 26303 attenuated arterial narrowing after SAH in both the prevention and reversal protocols. The protective effect of CGS 26303 achieved statistical significance at all dosages in the prevention protocol and at 30 mg/kg in the reversal protocol. These findings demonstrate that inhibiting the conversion of Big ET-1 to ET-1 via intravenous administration of an ECE inhibitor can be an effective strategy for limiting angiographic vasospasm after SAH. Moreover, the results demonstrate that treatment with the ECE inhibitor is capable of reducing vasospasm even when initiated after the process of arterial narrowing has begun. Finally, the results provide further support for the role of ET in the establishment of cerebral vasospasm. The ECE inhibitor CGS 26303 thus represents a promising therapeutic agent for the treatment of cerebral vasospasm following aneurysmal SAH.

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Barbara Cappelletto, Hakan H. Caner, Frank Schottler, Aij-Lie Kwan, David Eveleth, Patricia L. Foley, Neal F. Kassell and Kevin S. Lee

Calcium-activated proteolysis mediated by the protease inhibitor, calpain, has recently been implicated in the pathogenesis of cerebral vasospasm. The effect of one inhibitor of calcium-activated proteolysis, z-Leu-Phe-CONH-morpholene (zLF), on cerebrovascular constriction was examined in two experimental paradigms. In the first paradigm, the rabbit basilar artery (BA) was visualized via a transclival exposure, and its diameter was monitored using videomicroscopy. In the second experimental paradigm two intracisternal injections of autologous blood were administered to mimic a subarachnoid hemorrhage (SAH). The BA was visualized via the transclival exposure, and its luminal diameter was measured. Topical application of oxyhemoglobin (OxyHb), a known pathogenic agent in cerebral vasospasm, elicited vasoconstriction in normal animals, reducing arterial diameter to approximately 75% of resting levels. Pretreatment with zLF (100, 200, or 300 μM) attenuated vasoconstriction induced by OxyHb. In an experimental model of SAH, the diameter of the BA was reduced after the first injection of blood to approximately 67% of normal resting levels when measured 3 to 4 days later. This vasospastic response was reversed significantly by topical application of zLF (100 μM); vascular diameter was increased to approximately 84% of normal resting levels.

These findings demonstrate that both acute OxyHb-induced constriction and blood-induced vasospasm are sensitive to an inhibitor of the proteolytic enzyme, calpain. Together, these observations indicate an important role for calcium-activated proteolysis in the development and maintenance of vasospasm after SAH. In addition, it may be inferred from the data that inhibitors of calcium-activated proteolysis may be useful therapeutic agents for treating this form of cerebrovascular disease.

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Chih-Lung Lin, Aaron S. Dumont, Yu-Feng Su, Yee-Jean Tsai, Jih-Hui Huang, Kao-Ping Chang, Shen-Long Howng, Aij-Lie Kwan, Neal F. Kassell and Cheng-Hsing Kao


Cerebral vasospasm remains a major complication in patients who have suffered a subarachnoid hemorrhage (SAH). Previous studies have shown that 17β-estradiol (E2) attenuates experimental SAH–induced cerebral vasospasm. Moreover, E2 has been shown to reduce neuronal apoptosis and secondary injury following cerebral ischemia. Adenosine A1 receptor (AR-A1) expression is increased following ischemia and may represent an endogenous neuroprotective effect. This study was designed to evaluate the efficacy of E2 in preventing cerebral vasospasm and reducing secondary injury, as evidenced by DNA fragmentation and AR-A1 expression, following SAH.


A double-hemorrhage model of SAH in rats was used, and the degree of vasospasm was determined by averaging the cross-sectional areas of the basilar artery 7 days after the first SAH. A cell death assay was used to detect apoptosis. Changes in the protein expression of AR-A1 in the cerebral cortex, hippocampus, and dentate gyrus were compared with levels in normal controls and E2-treated groups (subcutaneous E2, 0.3 mg/ml).


The administration of E2 prevented vasospasm (p < 0.05). Seven days after the first SAH, DNA fragmentation and protein levels of AR-A1 were significantly increased in the dentate gyrus. The E2 treatment decreased DNA fragmentation and prevented the increase in AR-A1 expression in the dentate gyrus. There were no significant changes in DNA fragmentation and the expression of AR-A1 after SAH in the cerebral cortex and hippocampus in the animals in the control and E2-treated groups.


The E2 was effective in attenuating SAH-induced cerebral vasospasm, decreasing apoptosis in the dentate gyrus, and reducing the expression of AR-A1 in the dentate gyrus after SAH. Interestingly, E2 appears to effectively prevent cerebral vasospasm subsequent to SAH as well as attenuate secondary injury by reducing both apoptosis and a compensatory increase in AR-A1 expression in the dentate gyrus.

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Chih-Lung Lin, Aij-Lie Kwan, Aaron S. Dumont, Yu-Feng Su, Neal F. Kassell, Chih-Jen Wang, Shu-Chuan Wu, Ching-Ling Kuo, Ching-Shan Huang, Arco Y. Jeng and Chin-San Liu


Adhesion molecules, including intercellular adhesion molecule–1 (ICAM-1), vascular cell adhesion molecule–1 (VCAM-1), and E-selectin, are important mediators of inflammation, and their levels are elevated in the serum of patients following aneurysmal subarachnoid hemorrhage (SAH). The investigators previously found that CGS 26303 is effective in preventing and reversing arterial narrowing in a rabbit model of SAH. The purpose of the present study was to examine whether levels of adhesion molecules are altered after treatment with CGS 26303 in this animal model.


New Zealand White rabbits were each injected with 3 ml of autologous blood in the cisterna magna, and intravenous treatment with CGS 26303 (30 mg/kg) was initiated 1 hour later. The compound was subsequently administered at 12, 24, and 36 hours post-SAH. Blood samples were collected at 48 hours post-SAH to measure ICAM-1, VCAM-1, and E-selectin levels. After the rabbits had been killed by perfusion–fixation, the basilar arteries (BAs) were removed and sliced, and their cross-sectional areas were measured.

Treatment with CGS 26303 attenuated arterial narrowing after SAH. Morphologically, corrugation of the internal elastic lamina of BAs was prominently observed in the SAH only and vehicle-treated SAH groups, but not in the CGS 26303–treated SAH group or in healthy controls. There were no significant differences in the levels of VCAM-1 among the four groups. The levels of E-selectin were increased in all animals subjected to SAH (those in the SAH only, SAH plus vehicle, and SAH plus CGS 26303 groups) compared with healthy controls (no SAH); however, the levels of ICAM-1 in the SAH only and SAH plus vehicle groups were significantly elevated (p < 0.001), and treatment with CGS 26303 reduced ICAM-1 to control levels following SAH.


These results show that ICAM-1 may play a role in mediating SAH-induced vasospasm and that a reduction of ICAM-1 levels after SAH may partly contribute to the antispastic effect of CGS 26303.