Administration of a receptor antagonist for platelet-activating factor during equine endotoxaemia

Platelet-activating factor (PAF) is an important mediator of endotoxaemia and various PAF receptor antagonists prevent many of the adverse effects of experimental endotoxaemia in laboratory animals. In this study a specific PAF receptor antagonist was used to investigate the role of PAF in equine endotoxaemia. At an interval of not greater than 10 days, 6 horses were each challenged with endotoxin and endotoxin with concurrent administration of SRI 63-441, a PAF receptor antagonist. The order of the treatments was randomised. Clinical signs, serum biochemical and coagulation profiles, and platelet aggregation in vitro were monitored in all horses for 24 h after treatment. Challenge with endotoxin increased maximal platelet aggregation induced by PAF. This response was blocked by administration of SRI 63-441 concurrently with endotoxin. No changes in percentage maximal platelet aggregation to ADP or collagen were noted after administration of endotoxin. The PAF receptor antagonist delayed the onset of fever, tachycardia, leucopenia and lactic acidaemia. Lack of more profound beneficial alterations of the horses' responses to endotoxin may have been due to the low dose of endotoxin administered in this model or to only partial effectiveness of SRI 63-441 in blocking the effects of endotoxin-induced PAF.     

The platelet-activating factor antagonist BN 52021 attenuates hypoxic-ischemic brain injury in the immature rat

Platelet-activating factor (PAF) is overproduced in ischemic brain. Although postischemic PAF antagonist administration protects the mature brain in some models, little is known about the effects of PAF antagonists in the immature brain. We hypothesized that the PAF antagonist BN 52021 would attenuate perinatal cerebral hypoxic-ischemic injury. To elicit focal hypoxic-ischemic brain injury, 7-d-old (P7) rats (n = 111) underwent right carotid ligation, followed by 2.5-3.25 h of hypoxia (fractional concentration of inspired O2 = 0.08). BN 52021 neuroprotection was evaluated in three groups of experiments: 1) 25 mg/kg/dose, 0 and 2 h posthypoxia; 2), 25 mg/kg/dose immediately before and 1 h after hypoxia; and 3) posthypoxia-ischemia treatment with BN 52021 12.5, 25, or 50 mg/kg/dose in 2 doses 0 and 2 h after hypoxia. All experiments included concurrent vehicle-injected controls. To quantitate severity of injury, bilateral regional cross-sectional areas (groups 1 and 2) or hemisphere weights (group 3) were evaluated on P12. Both pre- and posthypoxic treatment with BN 52021 (25 mg/kg/dose, two serial doses) decreased the incidence of cerebral infarction from 90% to about 30% (p < 0.02, Fisher's exact test). Measurement of cross-sectional areas confirmed neuroprotection and indicated some benefit of pre- over posthypoxic-ischemic treatment in hippocampus and cortex. Over the dose range tested, the neuroprotective effect of BN 52021 administration was not dose-dependent. In contrast, BN 52021 did not attenuate N-methyl-D-aspartate-induced hippocampal excitotoxic injury in P7 rats. Either prophylactic or "rescue" administration of PAF antagonists decreases the incidence and severity of brain injury associated with an episode of perinatal cerebral hypoxia-ischemia.     

Purification and characterization of rhesus monkey liver amido hydrolases and their roles in the metabolic polymorphism for E6123, a platelet-activating factor receptor antagonist

We previously showed that a polymorphism for E6123 [(S)-(+)-6- (2-chlorophenyl)-3-cyclopropanecarbonyl-8,11-dimethyl-2,3,4,5- tetrahydro-8H-pyrido[4',3':4,5]thieno[3,2-f][1,2,4]triazolo[4,3-a] [1,4]diazepine] metabolism exists only in rhesus monkeys. In the present study, we purified, from rhesus monkey hepatic microsomes, three amido hydrolases that are involved in the metabolic polymorphism. Two forms of amido hydrolase from an extensive metabolizer and one from a poor metabolizer were purified by Q-Sepharose Fast Flow, Red A-agarose, octylamino-Sepharose 4B, and hydroxyapatite-Ultrogel chromatography, after solubilization with Lubrol. The three purified enzymes had the same molecular mass (47 kDa), and their amino-terminal amino acid sequences were identical. The enzymes were different from various known carboxylesterases in terms of substrate specificity, molecular mass, and amino-terminal amino acid sequence. They resembled arylacetamide deacetylase from human hepatic microsomes with respect to molecular mass and amino-terminal amino acid sequence. The KM values of the high and low affinity enzymes in the extensive metabolizer and the sole enzyme in the poor metabolizer were 37.6, 73.0, and 76.5 microM, respectively. The Vmax values were 3312.4, 504.8, and 427.9 pmol/min/mg of protein, respectively. The high affinity enzyme in extensive metabolizer appears to be quite distinct, whereas the low affinity enzyme in extensive metabolizer in similar or identical to the sole enzyme in poor metabolizer. Thus, the metabolic polymorphism in rhesus monkey may depend upon the existence of the high affinity enzyme in extensive metabolizer.     

Effects of a new platelet-activating factor antagonist, UR-12670, on several endotoxic shock markers in rats

Chemical and pharmaceutical research have provided physicians with an array of drugs that have beneficial effects on a variety of diseases. Such drugs, however, mostly help in controlling the manifestations of the pathological condition but do not permanently modify the underlying cause. Hence the necessity of new forms of therapy that change drastically the current approach to medical treatment. Gene therapy, with its potential to correct the malfunctioning genes at the origin of variety of diseases, seems to fulfill the requirements of this therapeutic "revolution". The feasibility of such an approach is underscored by the improved knowledge of the molecular mechanism and/or gene defects at the origin of acquired diseases widely spread in the population, and of more rare congenital conditions. The technical advances in molecular biology and genetic engineering achieved in the last ten years, offer the tools necessary to implement such therapeutic interventions. Here we present the approaches currently employed for gene therapy in the context of recent clinical trials. The scientific, ethical and economical implications deriving from a prospective routine use of gene therapy in the clinical setting are discussed.     

Pathogenesis of hyponatremia following subarachnoid hemorrhage due to ruptured cerebral aneurysm

BACKGROUND: Hyponatremia following subarachnoid hemorrhage (SAH) occurs due to the inappropriate secretion of antidiuretic hormone (SIADH). However, this condition is also sometimes associated with certain dehydration states. METHODS: To clarify the pathogenesis, daily values of urine volume, water balance, and sodium balance (Na Bal) were correlated with plasma levels of atrial natriuretic peptide (ANP), antidiuretic hormone (ADH), and plasma renin activity (PRA) in 31 cases of SAH. RESULTS: Na Bal was markedly negative on days 2 and 3. Cumulative Na Bal showed continuous negative values until day 10 following SAH. ANP values showed a consistent elevation, while ADH showed only an initial surge. PRA, as the gross indicator of circulatory volume, showed a lack of suppression, indicating no increase in the circulatory volume. CONCLUSION: Hyponatremia following SAH therefore appears to be the result of increased natriuresis, due to the inappropriate elevation of ANP rather than SIADH. In this situation, water restriction should not be recommended, since the circulatory volume is decreased.     

Effect of Y-24180, an antagonist at platelet-activating factor (PAF) receptors, on IgE-mediated cutaneous reactions in mice

OBJECTIVE AND DESIGN: We examined the effect of Y-24180, a potent platelet-activating factor (PAF) antagonist, on IgE-mediated cutaneous reactions in mice. MATERIALS: Female BALB/c mice were used. TREATMENT: Drugs were orally administered 1 h before a dinitrofluorobenzene (DNFB) challenge or 1 h before and 12 h after the challenge. METHODS: Biphasic increase in ear thickness, with peak responses at 1 h (immediate phase reaction, IPR) and 24 h (late phase reaction, LPR) after the DNFB challenge, were induced in mice which had been passively sensitized with monoclonal anti-dinitrophenyl IgE antibody 24 h before the DNFB challenge. Ear thickness was measured with a dial thickness gauge. RESULTS: Y-24180, WEB2086, ketotifen, and suplatast suppressed the IPR. Y-24180 also suppressed the LPR when administered once at 10 mg/kg or twice at 1 to 10 mg/kg. WEB2086 suppressed the LPR only when administered twice. However, ketotifen and suplatast did not suppress the LPR even when administered twice. Single administration of prednisolone significantly suppressed both the IPR and LPR. CONCLUSIONS: These results indicate that PAF may be involved in the induction of biphasic cutaneous reactions mediated by IgE, and Y-24180 is more effective compared with WEB2086 in this model. It is possible that the difference in the effectiveness between Y-24180 and WEB2086 depends on the persistence of those activities.     

Endothelial injury following experimental subarachnoid hemorrhage in rats: effects on brain blood flow

BACKGROUND: The leading cause of death and disability in patients suffering from aneurysmal subarachnoid hemorrhage (SAH) is cerebral vasospasm, a persistent, progressive, and often irreversible constriction of cerebral arteries. A wide array of pathological changes occur in cerebral arteries following SAH, with endothelial injury being the earliest and most consistent one. Since intact endothelium modulates many reflexes that influence vascular tone, damage to them may represent a significant contributor to cerebral vasospasm. METHODS: Changes in local cerebellar blood flow (LCBF) and pathological alterations in major cerebral arteries were studied and compared in rats at various time intervals following SAH. SAH induced by the subarachnoid injection of 0.3 ml of whole blood. Sham rats received a subarachnoid injection of 0.3 ml of isotonic saline. RESULTS: Except for an immediate but transient decrease, LCBF remained unchanged over a 3 day period following saline injection. Likewise, there were no pathological alterations in cerebral arteries of saline-injected rats. In contrast, the subarachnoid injection of whole blood produced significant changes in both LCBF and cerebral arteries. Within 30 minutes post-blood injection, LCBF became significantly decreased and remained so for 4 hours. However, within 24 hours, LCBF had returned to control levels where it remained for 3 days. Endothelial injury was observed in the basilar and middle cerebral arteries from 30 minutes through 4 hours, the same periods in which LCBF was significantly reduced. Within 24 hours, the time period in which LCBF had rebounded to control ranges, cerebral arteries showed no evidence of endothelial damage and resembled control cells. CONCLUSION: The results indicate a direct correlation between changes in LCBF and the structural integrity of endothelial cells in the early stages following SAH. The lack of chronically depressed LCBF (after 1 day) may be related to the quick structural repair of endothelium.     

Effect of a platelet-activating factor (PAF) antagonist, SR 27417A, on PAF-induced gas exchange abnormalities in mild asthma

Chelonacarus elongatus n. gen., n. sp. is proposed for a cheyletoid mite (Acari: Prostigmata) of the family Cloacaridae found in the cloacal tissue of the endangered green turtle Chelonia mydas Linnaeus, 1758 from the Atlantic coast of the Republic of Panama. In females, the new genus is distinguished from other genera of turtle cloacarids by the elongate slender shape of the idiosoma, the shape and pattern of sclerotization of the dorsal shield, and the fused distal ends of apodemes II. A combination of other features that distinguish the newly proposed genus is the smooth surface of the pedipalps, single dorsal spine on tibiae I-IV, no setae on coxa IV, terminal position of the vulva, and the strongly developed pair of ventral spines on tarsi I-II. This is the first record of cloacarids from sea turtles. The similarity of adult cloacarids in the genus Chelonacarus from sea turtles (Chelonioidea) and Cloacarus Camin et al., 1967 from snapping turtles (Chelydridae) lends support to the hypothesis of some paleontologists that these 2 groups of turtles are linked phylogenetically.     

Elevation of platelet activating factor, inflammatory cytokines, and coagulation factors in the internal jugular vein of patients with subarachnoid hemorrhage

The hemodynamic responses to acute (45 min) partial aortic constriction were studied in conscious intact (N = 7) or sinoaortic denervated (SAD) adult male Wistar rats (280-350 g, N = 7) implanted with carotid and femoral arterial catheters, a pneumatic cuff around the abdominal aorta and a pulsed Doppler flow probe to measure changes in aortic resistance. In addition, the hypertensive response and the reflex bradycardia elicited by total (N = 8) vs partial (N = 7) aortic constriction (monitored by maintenance of the pressure distal to the cuff at 50 mmHg) were compared in two other groups of intact rats. Intact rats presented a smaller hypertensive response (26 to 40% above basal level) to partial aortic constriction than SAD rats (38 to 58%). The calculated change in aortic resistance imposed by constriction of the aorta increased progressively only in intact rats, but was significantly smaller (193 to 306%) than that observed (501 to 591%) in SAD rats. Intact rats showed a significant bradycardia (23 to 26% change in basal heart rate) throughout coarctation, whereas the SAD rats did not (1 to 3%). Partial or total occlusion of the aorta induced similar hypertensive responses (37-38% vs 24-30% for total constriction) as well as reflex bradycardia (-15 to -17% vs -22 to -33%) despite a greater gradient in pressure (97-98 vs 129-140 mmHg) caused by total constriction. The present data indicate that the integrity of the baroreflex in intact rats can cause the hypertensive response to level off at a lower value than in SAD rats despite a progressive increase in aortic resistance. In addition, they also indicate that the degree of partial aortic constriction by maintenance of the pressure distal to the cuff at 50 mmHg already elicits a maximal stimulation of the arterial baroreflex.     

Stimulation of platelet-activating factor synthesis by neurotransmitters in salivary glands

Platelet-activating factor (PAF), a phospholipid mediator exhibiting potent biological activities, has been shown to stimulate amylase release from the pancreas and salivary glands. The capacity of salivary glands for PAF biosynthesis in response to stimulation has also been demonstrated. To elucidate the role of PAF in salivary glands, we studied the regulation of platelet-activating factor synthesis by the autonomic nervous system in canine salivary glands. Acetylcholine and ionomycin stimulated PAF production in dispersed cells from parotid, submandibular, and sublingual glands of dogs. Norepinephrine and phenylephrine, but not isoproterenol, also stimulated PAF production in submandibular gland cells. Norepinephrine-induced PAF production was blocked by phentolamine but not by propranolol. Acetylcholine and norepinephrine increased both the PAF production and liberation of [14C]arachidonic acid from cells pre-labeled with [14C]arachidonic acid in the presence of Ca2+ in the medium. These stimulants increased [14C]arachidonic acid liberation without the accompanying production of PAF in Ca(2+)-deprived medium. No activators or inhibitors of protein kinase C produced or affected acetylcholine-induced PAF production. Lyso-PAF:acetyl-CoA acetyltransferase was activated in the cells treated with acetylcholine, norepinephrine, isoproterenol, and 8Br-cyclic AMP. Deprivation of Ca2+ in the medium markedly reduced acetylcholine-induced activation of the transferase, but little affected norepinephrine-, isoproterenol-, and 8Br-cyclic AMP-induced activation. Dithiothreitol-insensitive cholinephosphotransferase activity was also increased by acetylcholine, norepinephrine, isoproterenol, and 8Br-cyclic AMP, and the deprivation of Ca2+ in the medium further increased the activation of the enzyme activity by these agents. These results suggest that PAF synthesis in canine salivary glands is under the control of muscarinic cholinergic and alpha-adrenergic systems via Ca(2+)-dependent remodeling pathways, and that the independent activation of either phospholipase A2 or acetyltransferase is insufficient for PAF production in submandibular gland cells, i.e., the concurrent activation of these enzymes is required.     

Myocardial stunning in the context of a subarachnoid hemorrhage

Myocardial stunning has been poorly described in patients with cerebrovascular accidents. We present a patient in whom severe anteroapical wall motion abnormalities and extensive anterior ST-segment elevation developed after subarachnoid hemorrhage. Total recovery ensued within 2 days. Coronary vasospasm induced by stroke-related sympathetic surge might be the determinant factor of this cardiac event.     

Monoclonal antibodies against ICAM-1 and CD18 attenuate cerebral vasospasm after experimental subarachnoid hemorrhage in rabbits

BACKGROUND AND PURPOSE: Inflammatory responses have been implicated in the elaboration of several forms of central nervous system injury, including cerebral vasospasm after subarachnoid hemorrhage (SAH). A critical event participating in such responses is the recruitment of circulating leukocytes into the inflammatory site. Two of the key adhesion molecules responsible for the attachment of leukocytes to endothelial cells are intercellular adhesion molecule-1 (ICAM-1) and the common beta chain of the integrin superfamily (CD18). This study examined the effects of monoclonal antibodies on ICAM-1 and the effects of CD18 on cerebral vasospasm after SAH. METHODS: A rabbit model of SAH was utilized to test the influence of intracisternally administered antibodies to ICAM-1 and CD18 on cerebral vasospasm. Antibodies were administered alone or in combination, and the cross-sectional area of basilar arteries was assessed histologically on day 2 post-SAH. RESULTS: Treatment with antibodies to ICAM-1 or CD18 inhibited vasospasm by 22% and 27%, respectively. When administered together, the attenuation of vasospasm increased to 56%. All of these effects achieved statistical significance. CONCLUSIONS: These findings provide the first evidence that the severity of cerebral vasospasm can be attenuated using monoclonal antibodies against ICAM-1 and CD18. The results reinforce the concept that cell-mediated inflammation plays an important role in cerebral vasospasm after SAH and suggest that therapeutic targeting of cellular adhesion molecules can be of benefit in treating cerebral vasospasm.     

Effect of transluminal angioplasty on cerebral blood flow in the management of symptomatic vasospasm following aneurysmal subarachnoid hemorrhage

In this study the authors have examined the effects of transluminal angioplasty on cerebral blood flow (CBF) in the management of intractable vasospasm following aneurysmal subarachnoid hemorrhage (SAH). Fourteen consecutively enrolled patients underwent attempted angioplasty with or without intraarterial infusion of papaverine. Twelve patients underwent pre- and postangioplasty xenon-enhanced computerized tomography (Xe-CT) scanning to measure regional CBF in 55 to 65 regions of interest (ROIs) per patient. Angioplasty was possible in 13 (93%) of 14 patients, with angiographically demonstrated improvement in all 13. Twelve (92%) of the 13 patients were neurologically improved following angioplasty; seven (58%) of the 12 patients who improved had a complete reversal of all delayed ischemic deficits. Angioplasty significantly decreased the mean number of ROIs at risk (11.4 ROIs pre- and 0.9 ROIs postangioplasty) (p < 0.00005, t-test). All patients had a reduction in the number of ROIs at risk after angioplasty; six (50%) of 12 no longer had any ROIs remaining at risk after angioplasty. Angioplasty significantly increased the mean CBF within at-risk ROIs (13 ml/100 g/minute pre- and 44 ml/100 g/minute postangioplasty) (p < 0.00005, t-test). All patients experienced an improvement in mean CBF in at-risk ROIs after angioplasty, with the mean CBF improving to above 20 ml/100 g/minute in all cases. No differences in the degree of improvement were found in patients who received intraarterial papaverine compared with those who did not. In the majority of patients with refractory vasospasm following SAH, angioplasty effectively dilated spastic arteries, reversed delayed neurological deficits, and significantly improved CBF in areas of brain at risk of infarction.     

The effect of a free radical scavenger and platelet-activating factor antagonist on FFA accumulation in post-ischemic canine brain

The effects of the platelet-activating factor antagonist BN 50739 and a free radical scavenger dimethyl sulfoxide on the accumulation of free fatty acids in post-ischemic canine brain are reported. Following 14 min of complete normothermic ischemia and 60 min of reperfusion, the total brain FFAs were approximately 150% higher than in the control group (p < 0.05). Perfusion with the platelet-activating factor antagonist BN50739 in its diluent dimethyl sulfoxide during 60 min of post-ischemic reoxygenation resulted in a 61.8% (p < 0.01) reduction in the total brain free fatty acid accumulation. Palmitic, stearic, oleic, linoleic, and arachidonic acids decreased by 53.8%, 63.5%, 69.0%, 47.4%, and 57.2%, respectively. Although dimethyl sulfoxide alone caused stearic and arachidonic acids to return to the normal concentration range, BN 50739 had a significant influence on recovery of palmitic, oleic, and linoleic acids and was previously shown to provide significant therapeutic protection against damage to brain mitochondria following an ischemic episode. Because free fatty acid accumulation is one of the early phenomena in cerebral ischemia, this study provides evidence to support the hypothesis that both platelet-activating factor and free radicals are involved in initiating cerebral ischemic injury.     

Reduction of microtubule catastrophe events by LIS1, platelet-activating factor acetylhydrolase subunit

Forming the structure of the human brain involves extensive neuronal migration, a process dependent on cytoskeletal rearrangement. Neuronal migration is believed to be disrupted in patients exhibiting the developmental brain malformation lissencephaly. Previous studies have shown that LIS1, the defective gene found in patients with lissencephaly, is a subunit of the platelet-activating factor acetylhydrolase. Our results indicated that LIS1 has an additional function. By interacting with tubulin it suppresses microtubule dynamics. We detected LIS1 interaction with microtubules by immunostaining and co-assembly. LIS1-tubulin interactions were assayed by co-immunoprecipitation and by surface plasmon resonance changes. Microtubule dynamic measurements in vitro indicated that physiological concentrations of LIS1 indeed reduced microtubule catastrophe events, thereby resulting in a net increase in the maximum length of the microtubules. Furthermore, the LIS1 protein concentration in the brain, measured by quantitative Western blots, is high and is approximately one-fifth of the concentration of brain tubulin. Our new findings show that LIS1 is a protein exhibiting several cellular interactions, and the interaction with the cytoskeleton may prove to be the mode of transducing a signal generated by platelet-activating factor. We postulate that the LIS1-cytoskeletal interaction is important for neuronal migration, a process that is defective in lissencephaly patients.     

Oxidative stress in the human brain after subarachnoid hemorrhage

OBJECT: The aim of this study was to verify the patterns of antioxidant enzymatic activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the human brain after subarachnoid hemorrhage (SAH) to verify whether an "oxidative stress situation" characterizes the brain response to subarachnoid bleeding. METHODS: Forty samples of gyrus rectus or temporal operculum that were obtained during a surgical approach to anterior circulation aneurysms were used for this study. The activity of total SOD, GSH-Px, and the SOD/GSH/Px ratio (which expresses the balance between the production of hydrogen peroxides by dismutation of superoxide radicals and the scavenging potential) were calculated in each case. Twelve samples were obtained from patients who underwent surgery for unruptured aneurysms (control group); 13 samples were obtained during surgical procedures performed within 72 hours of SAH; and 15 samples were obtained during delayed surgical procedures (> 10 days post-SAH). Ten patients presented with clinical deterioration caused by arterial vasospasm. In both SAH groups, the mean total SOD activity was significantly higher than in the control group (p=0.029). The mean activity of GSH-Px did not differ significantly between the SAH and control groups (p=0.731). There was a significant increase in the SOD/GSH-Px ratio in both SAH groups, as compared with controls (p < 0.05). There was a significant correlation between the enzymatic activity and the clinical severity of the hemorrhage, with findings of lower values of SOD and, mainly, of the SOD/GSH-Px ratio in the poor-grade patients. The SOD/GSH-Px ratio was 2.14+/-0.44 in patients who presented with clinical vasospasm and 1.24+/-0.2 in cases without vasospasm. CONCLUSIONS: The results of this study show an imbalance of the antioxidant enzymatic activities in the human brain after SAH. which is linked to the severity of the initial bleeding and possibly modified by the development of arterial vasospasm.