SAH-only Group Research Articles

Prevention of cerebral vasospasm by local delivery of cromakalim with a biodegradable controlled-release system in a rat model of subarachnoid hemorrhage

One mechanism that contributes to cerebral vasospasm is the impairment of potassium channels in vascular smooth muscles. Adenosine triphosphate-sensitive potassium channel openers (PCOs) appear to be particularly effective for dilating cerebral arteries in experimental models of subarachnoid hemorrhage (SAH). A mode of safe administration that provides timed release of PCO drugs is still a subject of investigation. The authors tested the efficacy of locally delivered intrathecal cromakalim, a PCO, incorporated into a controlled-release system to prevent cerebral vasospasm in a rat model of SAH. Cromakalim was coupled to a viscous carrier, hyaluronan, 15% by weight. In vitro release kinetics studies showed a steady release of cromakalim over days. Fifty adult male Sprague-Dawley rats weighing 350-400 g each were divided into 10 groups and treated with various doses of cromakalim or cromakalim/hyaluronan in a rat double SAH model. Treatment was started 30 minutes after the second SAH induction. Animals were killed 3 days after treatment, and the basilar arteries were processed for morphometric measurements and histological analysis. Controlled release of cromakalim from the cromakalim/hyaluronan implant at a dose of 0.055 mg/kg significantly increased lumen patency in a dose-dependent manner up to 94 +/- 8% (mean +/- standard error of the mean) of the basilar arteries of the sham group compared with the empty polymer group (p = 0.006). Results in the empty polymer group were not different from those in the SAH-only group, with a lumen patency of 65 +/- 12%. Lumen patencies of the cromakalim-only groups did not differ in statistical significance at low (64 +/- 9%) or high (66 +/- 7%) doses compared to the SAH-only group. Treatment of SAH with a controlled-release cromakalim/hyaluronan implant prevented experimental cerebral vasospasm in this rat double hemorrhage model; this inhibition was dose-dependent. The authors' results confirm that sustained delivery of cromakalim perivascularly to cerebral vessels could be an effective therapeutic strategy in the treatment of cerebral vasospasm after SAH.

Effects of gap junctional blockers on cerebral vasospasm after subarachnoid hemorrhage in rabbits

The role of gap junctional communication in the coordination of vascular behavior has been well established experimentally. The aim of this study was to investigate whether the gap junctional blockers would inhibit cerebral vasospasm after experimental subarachnoid hemorrhage (SAH). We used the double-hemorrhage model of SAH with injection of autologous arterial blood into the cisterna magna on days 1 and 2. Octanol or carbenoxolone was administered intracisternally on day 3, and repeated on days 4 and 5. Angiography was performed before (day 0) and 7 days after (day 7) SAH, and the diameter of the basilar artery was measured. Paraffin blocks of brain tissues were prepared and sectioned for hematoxylin and eosin staining for morphologic analysis. Arterial narrowing in SAH + octanol group and SAH + carbenoxolone group at day 7 was significantly less than that in SAH-only group and each SAH + vehicle group, respectively. Less morphologic changes in SAH + octanol group and SAH + carbenoxolone group were observed when compared to that in SAH-only group and each SAH + vehicle group. Western blotting showed that carbenoxolone down-regulated C×43 protein expression in the BA, which in the SAH-only group was significantly higher than that of the normal group. Gap junction blockers attenuate the experimental cerebral vasospasm and down-regulate the increase in expression of C×43 protein after SAH in rabbits. These data suggest that gap junctions play an important role in the development of cerebral vasospasm.

Effects of raloxifene on cerebral vasospasm after experimental subarachnoid hemorrhage in rabbits

Background The aim of this study was to investigate the ability of a SERM, RLX, to prevent vasospasm in a rabbit model of SAH. Methods Thirty-four New Zealand white rabbits were allocated into 3 groups randomly. Subarachnoid hemorrhage was induced by injecting autologous blood into the cisterna magna. The treatment groups were as follows: (1) sham operated (no SAH [n = 12]), (2) SAH only (n = 12), and (3) SAH plus RLX (n = 10). Basilar artery lumen areas and arterial wall thickness were measured to assess vasospams in all groups. Results There was a statistically significant difference between the mean basilar artery cross-sectional areas and the mean arterial wall thickness measurements of the control and SAH-only groups ( P < .05). The difference between the mean basilar artery cross-sectional areas and the mean arterial wall thickness measurements in the RLX-treated group was statistically significant ( P < .05). The difference between the SAH group and the SAH + RLX group was also statistically significant ( P < .05). Conclusions These findings demonstrate that RLX has marked vasodilatatory effect in an experimental model of SAH in rabbits. This observation may have clinical implications suggesting that this SERM drug could be used as possible anti-vasospastic agent in patients without major adverse effects.

Inhibitory effect of gap junction blockers on cerebral vasospasm

The gap junction is important in the propagation of dilation/constriction signals along vessels for coordinated behavior in control of vascular tone. The authors hypothesized that gap junctions might play a role in cerebral vasospasm following subarachnoid hemorrhage (SAH). The aims of the present study were to investigate the role of gap junctions and to observe the potential therapeutic efficacy of gap junction blockers in cerebral vasospasm in vitro and in vivo. For the in vitro investigation, the effect of heptanol on the oxyhemoglobin (HbO(2))-induced contraction of isolated rabbit basilar arteries (BAs) was observed by using an isometric tension-recording method. For the in vivo experiments, the potential therapeutic efficacy of heptanol and carbenoxolone was surveyed after it was given intravenously in the rabbit double-hemorrhage model. Light microscopy was performed to assess the morphological changes in the arteries examined. For the in vitro method, heptanol significantly inhibited the sustained contraction induced both by HbO(2) and K(+) in the BA rings. The magnitude of the heptanol-induced relaxation was dose dependent. The inhibitory effect of heptanol on the K(+)-induced vasoconstriction was weaker than that on the HbO(2)-induced constriction. After arterial rings were pretreated for 10 minutes, heptanol significantly decreased their responses to the HbO(2)-induced contraction. For the in vivo method, heptanol and carbenoxolone significantly decreased the narrowing of BAs when given intravenously in the rabbit double-hemorrhage model. In both treated groups, the diameters of the arteries had not changed significantly on Day 7 compared with those of the arteries in the SAH + vehicle and the SAH-only group. Heptanol and carbenoxolone significantly inhibited the experimental cerebral vasospasm both in vitro and in vivo. Blockage of gap junctions is a probable candidate for a new approach in the treatment of cerebral vasospasm. Gap junctions may play a pathophysiological role in cerebral vasospasm.

Delayed Intracranial Delivery of a Nitric Oxide Donor from a Controlled-release Polymer Prevents Experimental Cerebral Vasospasm in Rabbits

Decreased local availability of nitric oxide (NO) may mediate chronic vasospasm after aneurysmal subarachnoid hemorrhage (SAH). Previous reports have shown that early treatment with NO prevents vasospasm in animals. We evaluated the efficacy of controlled-release polymers that contain the NO donor diethylenetriamine (DETA-NO) for the delayed treatment of vasospasm in a rabbit model of SAH. DETA-NO 20% (wt/wt) was incorporated into ethylene-vinyl acetate (EVAc) polymers. Animals (n = 52) were randomized to two experimental groups. In the first group (n = 32), animals received SAH and implantation of either 20% DETA-NO/EVAc polymer at a dose of 0.5 mg/kg of DETA-NO (n = 16) or empty EVAc polymer (n = 16). Polymers were implanted 24 (n = 16) or 48 hours (n = 16) after SAH. In the second group (n = 20), animals received SAH and implantation of either 20% DETA-NO/EVAc polymer at a dose of 1.3 mg/kg (n = 10) or empty EVAc (n = 10). Polymers were implanted 24 (n = 10) or 48 hours (n = 10) after SAH. An additional group (n = 16) underwent either sham operation (n = 6) or SAH only (n = 10). Animals were killed 3 days after hemorrhage, and the basilar arteries were processed for morphometric measurements. Results were analyzed using Student's t test. Treatment with 20% DETA-NO/EVAc polymers at a dose of 1.3 mg/kg significantly increased basilar artery lumen patency when administered at 24 (97 +/- 6% versus 73 +/- 10%; P = 0.0396) or 48 hours (94 +/- 6% versus 71 +/- 9%; P = 0.03) after SAH. Treatment with 20% DETA-NO/EVAc polymers at a dose of 0.5 mg/kg administered 48 hours after SAH significantly increased lumen patency (82 +/- 8% versus 68 +/- 12%; P = 0.03); a dose of 0.5 mg/kg, 24 hours after SAH, did not reach statistical significance (74 +/- 7% versus 65 +/- 9%; P = 0.16). The SAH-only group had a lumen patency of 67 +/- 12%. Delayed treatment of SAH with controlled-release DETA-NO polymers prevented experimental posthemorrhagic vasospasm in the rabbit. This inhibition was dose-dependent. This further confirms the role of NO in the pathogenesis of vasospasm.

Prevention of vasospasm by anti-CD11/CD18 monoclonal antibody therapy following subarachnoid hemorrhage in rabbits.

Adhesion of leukocytes and their migration into the periadventitial space may be critical in the pathophysiology of vasospasm following subarachnoid hemorrhage (SAH). The cell adhesion molecules involved in this process are lymphocyte function-associated antigen-1 (CD11a/CD18) and macrophage antigen-1 (CD11b/CD18), which are present on neutrophils/macrophages, and intercellular adhesion molecule-1 (CD54), which is present in endothelial cells. A humanized monoclonal antibody (mAb), Hu23F2G, targets CD11/CD18 and prevents leukocyte adhesion to endothelial cells. In this study, systemic administration of Hu23F2G prevented vasospasm in the rabbit model of SAH. Twenty-six New Zealand White rabbits were injected with autologous blood into the cisterna magna to induce SAH, after which they were randomized to receive injections of either Hu23F2G (10 animals) or a placebo at 30 minutes and 24 and 48 hours after SAH (six animals). Control animals underwent sham operations (four animals) or SAH alone (six animals). The animals were killed 72 hours after SAH, their bodies perfused and fixed, and their basilar arteries processed for morphometric analysis. Peripheral white blood cells (WBCs) were counted at 72 hours. The percentages of lumen patency were compared using the Student t-test. The presence of neutrophils and macrophages was confirmed by immunohistochemical analysis in which a rat anti-rabbit anti-CD18 mAb and cresyl violet were used. Treatment with Hu23F2G resulted in the significant prevention of vasospasm. Animals treated with Hu23F2G had 90 +/- 7% lumen patency compared with 65 +/- 7% in the placebo group (p = 0.025). The percentage of lumen patency in the SAH-only group was 59 +/- 10%. The mean WBC count was 16,300 +/- 2710/microl in the treatment group, compared with 7000 +/- 386/microl in the control group (p = 0.02). Administration of Hu23F2G produced increased numbers of WBCs in 70% of the animals treated. This study supports the concept that leukocyte-endothelial cell interactions play an important role in the pathophysiology of chronic vasospasm after SAH. Systemic therapy with an anti-CD11/CD18 mAb prevents vasospasm after SAH by inhibiting adhesion of neutrophils and macrophages and their migration into the periadventitial space.

Evaluation of functional response of cerebral arteries by a new morphometric technique

The contractile response of the rabbit basilar artery under four conditions was determined: (1) response in a resting condition without exclusion of the sympathetic nervous system (control group I); (2) response in a resting condition with α-adrenoceptor blockade (group II); (3) response to subarachnoid haemorrhage (SAH) (group III); and (4) response to SAH with α-adrenoceptor blockade (group IV). It was also ascertained whether it was possible to measure contractile response using a new morphometric method. Vessels were prepared by intracardial perfusion fixation, stained by haematoxylin and eosin, and the length of the intimal corrugations were measured by computer image analysis. Two procedures were followed in order to express the intensity of intimal corrugation, indicating the contractibility of the basilar arteries: (1) the corrugation coefficient (CC) of the basilar artery intima was estimated by dividing the precisely measured length of the intimal corrugations by the length of the measured vessel wall section of the vessel cross-sections (obtained histologically); (2) the lumen reduction coefficient (LRC) of the basilar artery was determined by dividing the “ideal” luminal area (calculated from the total length of the intimal circumference) by the real luminal cross-section area. The results of CC measurements revealed the smoothest intima (mean CC=1.146, P=0.00) and the least reduction of lumen (mean LRC=0.26, P=0.000) in group II (rabbits without SAH but with α-blocker phenoxybenzamine), and in group IV (SAH group of rabbits with α-blocker phenoxybenzamine) where the mean CC was 1.141 ( P=0.001) and the mean LRC was 0.33 ( P=0.002) in comparison with the SAH-only group III, pointing out the effectiveness of α-blockade even against SAH vasospastic stimuli. Control group I (without SAH and without treatment) showed a greater degree of corrugation in the intima and an increased reduction in the lumina than in groups II and IV, but still significantly less than in group III (mean CC=1.197, P=0.001, and mean LRC=0.40, P=0.028), thus demonstrating a certain resting tone of the basilar arteries (in an ideal situation, without any tone at all, the CC and LRC would be equal one). The highest degree of intimal corrugation and the greatest lumina reduction were discovered in the SAH-only group III (mean CC=1.374 and mean LRC=0.60). The differences among groups I, II and IV were insignificant. The results of this study suggest four conclusions: (i) the possibility of evaluating the functional response of rabbit cerebral arteries using this new morphometric technique; (ii) the adrenergic influence on resting tone of these arteries; (iii) the likely preventive role of an α-blockade on post-SAH vasospasm of basilar arteries in rabbits; and (iv) good comparability of the results of CC and LRC measurements with the angiographically estimated vessel diameters of other similar studies.

Inhibition of poly(ADP-ribose) polymerase attenuates cerebral vasospasm after subarachnoid hemorrhage in rabbits.

Poly(ADP-ribose) polymerase (PARP) is important in modulating inflammation, which has been implicated in cerebral vasospasm after subarachnoid hemorrhage (SAH). We investigated the role of PARP in vasospasm using 3-aminobenzamide (3-AB), a PARP inhibitor, in a rabbit model. Twenty-four New Zealand White rabbits were divided into 4 groups: (1) no treatment (control group, n=6); (2) blood injection without pretreatment (SAH-only group, n=6); (3) blood injection with pretreatment by vehicle (SAH+vehicle group, n=6); and (4) blood injection with pretreatment by 3-AB (SAH+3-AB group, n=6). We used the single-hemorrhage model of SAH, injecting autologous arterial blood into the cisterna magna. Angiography was performed before (baseline) and after (day 2) SAH, and the diameter of the basilar artery (BA) was measured. Animals were euthanatized after the second angiogram. After perfusion and fixation, the brains were cut into sections for hematoxylin and eosin and immunohistochemical staining for poly(ADP-ribosyl)ation. In the control group, there were no differences in the BA lumen caliber between baseline and day 2 (96.8+/-10.4%). Cerebral vasospasm in the SAH+3-AB group (88.2+/-6. 2%) was remarkably attenuated in comparison with that in the SAH-only group (64.9+/-8.0%) and the SAH+vehicle group (65.6+/-10. 8%). The BA in the SAH+3-AB group showed less corrugation of the tunica elastica interna than that in the SAH-only and SAH+vehicle groups. Staining for poly(ADP-ribosyl)ation was markedly inhibited in smooth muscle and adventitial cells of the BA in the SAH+3-AB group compared with other groups. Inhibiting ADP-ribosylation attenuates cerebral vasospasm after SAH in rabbits, and PARP activation may play an important role in the development of cerebral vasospasm.

Systemic administration of mexiletine for attenuation of cerebral vasospasm following experimental subarachnoid haemorrhage.

Mexiletine is a class Ib drug that is widely used to treat ventricular arrhythmias. This compound is mainly known as a sodium channel blocker, but studies have demonstrated that it can also activate ATP-sensitive K+ channels and block Ca2+ channels. Recent in vitro data from experiments on liposomes indicate that mexiletine is also a potent antioxidant. The unique activity profile of this drug raised the possibility that it might be of benefit in limiting cerebral vasospasm. Our first series of experiments assessed the effects of mexiletine on transclivally exposed rabbit basilar arteries. The arteries were treated with 50-mM KCl, 20-nM endothelin-1 (ET-1), or 100-microM lysophosphatidic acid (LPA) in the presence or absence of 400-mM mexiletine. Vasoconstriction caused by KCl, ET-1, and LPA was inhibited by mexiletine. In a second series of experiments, subarachnoid haemorrhage (SAH) was induced in rabbits by injecting 3-ml of autologous arterial blood into the cisterna magna. Forty-eight hours after SAH induction, transclivally exposed basilar arteries exhibited a spastic constriction that was partially reversed by topical application of 400-microM mexiletine. In a third set of experiments, mexiletine was administered orally at dosages of 80-, 20, and 5-mg/kg/day t.i.d., beginning 3 hours before SAH to study the prevention of vasospasm. In a separate group of animals, 80- and 20-mg/kg/day t.i.d. of mexiletine was administered 21 hours post-SAH induction, to study the reversal of vasoconstriction. Microscopic analysis of vessels from controls (no SAH), SAH-only, and SAH + mexiletine groups indicated there was 71.43% vascular constriction in the SAH-only group compared with controls. Considerable vasorelaxation was seen in the prevention study, in which average arterial cross-sectional areas were reduced by only 17.86% and 39.29% in the mexiletine 80- and 20-mg/kg/day groups, respectively, compared with controls (p < 0.001). Compared with controls, average arterial cross-sectional areas were reduced by 53.58% and 64.29% in the mexiletine 80- and 20-mg/kg/day reversal groups, respectively. Our findings indicate that mexiletine induces potent relaxation in cerebrovascular arteries contracted with various agents, and that it prevents and partially reverses SAH-induced vasoconstriction.

Histidine attenuates cerebral vasospasm in a rabbit model of subarachnoid hemorrhage

BACKGROUND Free radical generation following hemolysis of a subarachnoid blood clot is believed to be a key component in the development of cerebral vasospasm. Histidine, an essential amino acid with free radical scavenging characteristics, was examined for its effects on cerebral vasospasm. METHODS An experimental rabbit model of subarachnoid hemorrhage-induced vasospasm was used in which autologous arterial blood was injected into the cisterna magna. Basilar arteries were removed following perfusion-fixation two days after the injection of blood, and their cross-sectional luminal areas were measured using computerized image analysis. Rabbits received intravenous injections of L-histidine or vehicle starting 30 min prior to induction of subarachnoid hemorrhage (SAH), with additional injections given four times per day for the next 2 days. RESULTS The luminal area of arteries from animals treated with histidine (50 mg/kg/dose or 100 mg/kg/dose) were significantly larger than those from vehicle-treated animals. Relative to the SAH-only groups (mean cross-sectional area = 106.8 × 10 3 μm 2), vasoconstriction was attenuated by 31% in the low dose treatment group (180.0 × 10 3 μm 2) and by 52% in the high dose treatment group (227.4 × 10 3 μm 2). Mean luminal area of control basilar arteries was 340.5 × 10 3 μm 2. CONCLUSIONS These findings demonstrate that histidine reduces the amount of cerebral vasospasm occurring subsequent to experimental SAH. It is suggested that the free radical scavenging characteristics of histidine, particularly its ability to scavenge singlet oxygen, may be responsible for the reduction in vasospasm.