K. Hongo

Effect of the nonglucocorticoid 21-aminosteroid u74006f on experimental cerebral vasospasm

The present work was performed to establish whether the nonglucocorticoid, 21-aminosteroid, U74006F, could prevent the development of delayed cerebral vasospasm after experimental subarachnoid hemorrhage. The subarachnoid hemorrhage was produced by percutaneous injection of 4.5 mL of nonheparinized autologous blood into the cisterna magna of rabbits. U74006F (1 mg/kg) or placebo was injected intraperitoneally every 12 hours starting 12 hours prior to induction of hemorrhage for a total of six doses. The animals were sacrificed by perfusion fixation. The basilar artery was removed on day 2 and processed for morphometric analysis. Control/placebo and subarachnoid hemorrhage/placebo basilar artery diameters were 651.2 +/- 25.4 and 366.3 +/- 34.2 mu, respectively. Control/U74006F basilar artery diameters (669.8 +/- 21.8 mu) were not significantly different from that of the control/placebo group. U74006F treatment greatly minimized subarachnoid hemorrhage-induced reduction in mean luminal diameter (563.7 +/- 48.2 mu) (p less than 0.001). These results demonstrate considerable therapeutic promise for U74006F in the prevention of cerebral vasospasm.

Mechanism of enlargement of major cerebral collateral arteries in rabbits.

Major cerebral collateral arteries enlarge following bilateral ligation of the common and internal carotid arteries. The purpose of this investigation was to determine the relative contribution of cellular hypertrophy versus cellular hyperplasia to this vessel change in a morphometric analysis as well as the functional properties of remodeled vessels in an in vitro study. We assessed cell number and vessel dimensions by morphometric analysis of 16 perfusion-fixed rabbit basilar arteries. Results demonstrated significant increases in luminal diameter from 761 to 946 microns (p less than 0.01), medial cross-sectional area from 5.1 x 10(4) to 7.6 x 10(4) micron2 (p less than 0.005), smooth muscle cell volume from 9.19 x 10(5) to 1.44 x 10(6) micron3 (p less than 0.0005), and overall arterial length from 17.41 to 20.36 mm (p less than 0.005) in basilar arteries from the eight ligated rabbits compared with the eight sham-operated controls. Smooth muscle cell volume fraction and cell numerical density were unchanged whereas the number of cells per unit length of artery was increased significantly from 21.5 to 31.0 cells/micron (p less than 0.05). These data indicate that smooth muscle cell hyperplasia rather than hypertrophy contributes to increases in vessel mass. Functional properties of the basilar arteries from 10 ligated and 10 normal control rabbits were analyzed in vitro. Results showed increased contraction to potassium chloride (approximately 74%) (p less than 0.01) and increased sensitivity of smooth muscle to acetylcholine (p less than 0.05) while maximal relaxation was the same as control in the ligated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of subarachnoid hemorrhage on calcitonin gene-related peptide-induced relaxation in rabbit basilar artery.

An isometric tension measurement of ring segments was performed in the rabbit basilar and common carotid arteries in vitro to investigate the regional differences in the calcitonin gene-related peptide (CGRP)-induced vasodilation and the effect of subarachnoid hemorrhage on CGRP-induced vasodilation. CGRP elicited vasodilation of the rabbit basilar artery in a dose-dependent fashion when the artery was precontracted by 10(-5) M 5-hydroxytryptamine, whereas almost no relaxation occurred in the rabbit common carotid artery. The relaxation of the basilar artery was 64.03 +/- 1.85% at 3 x 10(-8) M CGRP, with an EC50 of 8.46 +/- 0.08. Two days after experimental subarachnoid hemorrhage, CGRP-induced relaxation of the rabbit basilar artery was 53.96 +/- 8.08% of the 10(-5) M 5-hydroxytryptamine-induced contraction, not significantly different from that of the basilar artery of the control rabbit. Our findings suggest that CGRP induces potent vasodilation in the rabbit basilar artery and that no impairment of vasodilation occurred after experimental subarachnoid hemorrhage. We speculate that CGRP may have therapeutic potential in cerebrovascular disease such as vasospasm after subarachnoid hemorrhage.

Comparison of intraluminal and extraluminal inhibitory effects of hemoglobin on endothelium-dependent relaxation of rabbit basilar artery.

To determine whether extraluminal or intraluminal hemoglobin inhibits endothelium-dependent relaxation, we measured the vascular responsiveness of rabbit basilar artery in an in vitro perfusion system and we performed immunohistochemical staining for hemoglobin. In the in vitro study, we applied agents from either the intraluminal or the extraluminal side of excised basilar arteries. KCl-induced contraction was the same with either application. Acetylcholine-induced maximal relaxations were 57.6 +/- 8.5% of the contraction induced by 10(-5) M 5-hydroxytryptamine for control, 3.3 +/- 0.3% for intraluminal, and 34.9 +/- 8.6% for extraluminal applications. Adenosine triphosphate-induced maximal relaxations were 64.2 +/- 4.1% of the contraction induced by 10(-5) M 5-hydroxytryptamine for control, 26.9 +/- 3.8% for intraluminal, and 42.2 +/- 6.0% for extraluminal applications. Hemoglobins inhibition of acetylcholine- and adenosine triphosphate-induced relaxation was significantly greater with intraluminal than with extraluminal application (p less than 0.05). The immunohistochemical study revealed hemoglobin in the outer layer of the smooth muscle and in the adventitia when 10(-5) M hemoglobin was applied extraluminally for 5 minutes, whereas hemoglobin was observed on the surface of the endothelial cells after intraluminal application. Our findings suggest that hemoglobin inhibits acetylcholine- or adenosine triphosphate-induced relaxation by binding to endothelium-derived relaxing factor (EDRF) and by inhibiting production of EDRF. Hemoglobins inhibitory effect on endothelium-dependent relaxation may be important in the pathogenesis of vasospasm after subarachnoid hemorrhage.

Muscarinic cholinergic receptors on the endothelium of human cerebral arteries

To characterize the muscarinic cholinergic receptors on the endothelium of human cerebral arteries, isometric tension measurement and receptor autoradiographic studies were performed. Acetylcholine (ACh) induced dose-dependent relaxation of human cerebral arteries precontracted by 10−5 M serotonin, with an EC50 of 1.9 ± 0.6 × 10−6 M (n = 7). The relaxation was abolished by 10−5 M hemoglobin. Autoradiography, using the muscarinic antagonist [3H]propylbenzilylcholine mustard, demonstrated the high density of muscarinic cholinergic receptors on the endothelial cells of human cerebral arteries, especially on the luminal surface of the endothelium. These findings suggest that ACh-induced relaxation mediated by muscarinic cholinergic receptors on the endothelium has a physiological function in human cerebral arteries.

Immunohistochemical demonstration of transient increase in prostaglandin F2-alpha after recirculation in global ischemic rat brains

SummaryThe changes in prostaglandin F2-alpha (PG F2α) staining over 3 days of recirculation in both fore-and hindbrains were studied. Five minutes of global ischemia was produced in 24 rats by Pulsinellis method with hypotension around 50 mm Hg of mean arterial blood pressure. Eight rats (including three pretreated with indomethacin) were recirculated for 5 min, three for 1 h, five for 2 h and five for 3 days. Five normal rats without occlusion of vessels served as controls. The brains were snap frozen. Ten-micrometer cryosections were stained for PG F2α by the indirect immunofluorescence method after fixation in carbodiimide and in Zambonis solution. Positive staining for PG F2α was noted in pial vessels in all normal and ischemic rats. Recirculated rats revealed the strongest reaction at 5 min after recirculation in blood vessels and in neuronal cytoplasm (especially in hippocampi and in Purkinje cells). The intensity of staining was markedly reduced after 1 h. Rats pretreated with indomethacin showed less increase in staining. The above results indicate that recirculation after ischemia produces a transient increase in PG F2α in blood vessels and neurons of both fore- and hindbrains.

Time course of the blood-arterial wall barrier disruption following experimental subarachnoid haemorrhage

SummaryThe time course of the blood-arterial wall barrier disruption following experimental subarachnoid haemorrhage (SAH) was studied in 24 rabbits. Animals with SAH received two successive blood injections through the cisterna magna. Horseradish peroxidase (HRP) was given intravenously 30 minutes before sacrifice to assess the integrity of the barrier. In the basilar arteries taken from animals that were sacrificed 4 days after the first SAH, HRP-reaction products were diffusely observed in the subendothelial space. Three weeks following the first SAH, permeation of HRP was still observed in half of the animals. However, in animals sacrificed 7 weeks after the first SAH, no permeation of HRP into the subendothelial space was noted. Opening of the interendothelial space seemed to be the major mechanism for HRP permeation into the subendothelial space rather than transendothelial vesicular transport. Disruption of the bloodarterial wall barrier in the major cerebral arteries following SAH may play a role in the pathogenesis of vasospasm.

Metabolic alterations in rabbit cerebral arteries caused by subarachnoid hemorrhage.

The effect of subarachnoid hemorrhage on metabolic rates in rabbit cerebral arteries was investigated by measuring adenosine triphosphate (ATP) content and L-lactate release. The mean +/- SEM ATP content was 0.38 +/- 0.02 mumol/g wet wt in control rabbit basilar arteries (n = 6). The ATP content decreased significantly to 0.17 +/- 0.02 mumol/g wet wt 2 days after experimental subarachnoid hemorrhage (n = 6), although only a slight decrease was detected in the basilar arteries 2 days after cisternal injection of the same amount of artificial cerebrospinal fluid. Hypoxia significantly decreased ATP content in the control basilar arteries to 0.26 +/- 0.04 mumol/g wet wt (n = 6). The same degree of hypoxia did not decrease ATP content in the basilar arteries after subarachnoid hemorrhage. Release of L-lactate was significantly higher from the arteries after subarachnoid hemorrhage than from the control arteries under both aerobic and hypoxic conditions. Our results indicate that subarachnoid hemorrhage induced an alteration of metabolic rates in rabbit cerebral arteries. The oxygen-requiring pathways to synthesize ATP may be important in control cerebral arteries; however, after experimental subarachnoid hemorrhage, the main pathway in the cerebral arteries may shift from oxygen-requiring pathways to an anaerobic glycolytic pathway.

Cerebrovascular Effects of Substance P After Experimental Subarachnoid Haemorrhage

SummaryThe vasoactive effects of substance P (SP), as well as the content of cyclic guanine monophosphate (cGMP), were determined in the rabbit basilar artery after subarachnoid haemorrhage (SAH).Out of 47 rabbits, 24 were subjected to a SAH, induced by injecting 5ml of autologous arterial blood into the cisterna magna; 23 were used as controls. In 20 animals (10 SAH and 10 controls), isometric tension recording of isolated rings of the basilar artery — dissected 2 days after SAH — was employed to assess the dosedependent vasodilatation to SP (10−10 to 10−6M) after precontraction with serotonin (10−8 to 10−5M). In 15 animals (8 SAH and 7 controls), the basal cGMP content was measured in the basilar artery 2 days after SAH. In the other 12 animals (6 SAH and 6 controls), the increase in cGMP content was measured in the basilar artery after a 10-minute incubation with SP (10−6M).SP caused significantly less dilatation in animals subjected to SAH than in controls, especially for concentrations between 10−9 and 10−6M (p < 0.001). The cGMP content in the arteries 2 days after SAH was significantly lower than in control arteries (31.5 ± 7.3 against 57.3 ± 4.3 pmoles/g tissue). In the preparations incubated with SP, the increase of cGMP was 440 ± 115% in the control arteries, and only 97 ± 30% in the arteries after SAH.It is concluded that the vasodilator activity of SP is significantly impaired after SAH. Moreover, the changes in cGMP content after SAH suggest a link between impaired vasoactive response to SP and decreased production of cGMP after SAH.

Effect of hypoxia on endothelium-dependent relaxation of canine and rabbit basilar arteries

SummaryAn important role of endothelium-dependent relaxation in the local regulation of vascular tone has been suggested. In the present study, the effect of hypoxia on endothelium-dependent relaxation was investigated in canine and rabbit basilar and in rabbit common carotid arteriesin vitro, using an isometric tension recording method. Hypoxia was introduced by changing the gas mixture in thein vitro chamber from 95% O2-5% CO2 to 95% N2-5% CO2. Thrombin and acetylcholine were used to induce endothelium-dependent relaxation.Thrombin at 0.1 and 1.0U/ml, respectively, caused dose-dependent relaxation of the canine basilar artery precontracted by 10−6M prostaglandin F2α. Acetylcholine also evoked dose-dependent relaxation of rabbit basilar and common carotid arteries precontracted by serotonin. Under hypoxic conditions, the relaxing effect of thrombin or acetylcholine decreased both in canine and in rabbit arteries, although it was not significant in rabbit basilar arteries.It has been postulated that following subarachnoid haemorrhage, diffusion of oxygen to the walls of the major cerebral arteries might be impaired by the subarachnoid clot. This could cause hypoxia of the arteries and contribute to vasospasm by suppressing endothelium-dependent relaxation, as well as by enhancing the contractile responses of the cerebral arteries to the vasoconstrictor agents in the bloody cerebrospinal fluid.