Beagle puppy model of intraventricular hemorrhage: ethamsylate studies

Intraventricular hemorrhage (IVH) remains a major problem of preterm neonates, and ethamsylate, an inhibitor of specific prostaglandin-synthetic enzymes has been demonstrated to prevent IVH in these patients. We have examined the effects of ethamsylate on newborn beagle pups who were, by randomized computerized design, assigned to four cells consisting of (a) either ethamsylate or saline pretreatment and (b) either insulted or not insulted with hemorrhagic hypovolemia/volume re-expansion. Prostaglandin levels were obtained prior to and thirty minutes following administration of the solutions and 14C iodoantipyrine autoradiography was performed for cerebral blood flow (CBF) determinations. Ethamsylate produced a significant decrease in the incidence of IVH in this model (p less than 0.05). Following drug administration, ethamsylate-pretreated pups had significant declines in thromboxane B2 and 6-keto PGF1 alpha levels, the major breakdown products of thromboxane A2 and prostacyclin. Although ethamsylate significantly lowered baseline CBF in all brain regions examined in insulted and non-insulted pups (p less than 0.05), in the drug-treated group it did not prevent the changes seen in CBF to the germinal matrix region which were detected in the saline-pretreated pups. Nor did it significantly blunt the blood pressure changes in response to the hemorrhagic hypovolemia/volume re-expansion insult found in the latter group of animals. In addition, only ethamsylate pretreated pups had marked hypotensive responses to the reperfusion phase of the insult. Although the diminution of baseline CBF may contribute to the prevention of neonatal IVH which this drug has been demonstrated to exhibit, ethamsylate may also act as a capillary stabilizing agent.     

Cardiac and coronary consequences of intracoronary platelet activating factor infusion in the domestic pig

In previous studies we have shown that platelet-activating factor (PAF) is a potent vasoactive substance with deleterious effects on coronary blood flow (CBF) and myocardial performance. The present student further investigates the effects of PAF during its sustained intracoronary infusion in the blood-perfused domestic pig (n=16). PAF infusion (1–9nmol/min) produced triphasic changes in CBF (n=7): an initial brief phase of coronary dilation (14 ± 2%) above baseline), followed by severe reduction in CBF due to increase in coronary vascular resistance and a third phase of escape that was characterized by return of CBF towards baseline in spite of continuing PAF infusion. In 9 remaining pigs PAF infusion had a biphasic response: the first phase of coronary dilation rapidly turned into severe coronary constriction accompanied by severe systemic hypotension and death within a few min. PAF infusion caused a profound rise in systemic arterial and coronary venous thromboxane B₂ levels, while 6-keto-PGF_1α and leukotriene C₄-immunoreactivity levels were not changed. Indomethacin completely blocked the rise in thromboxane level during PAF infusion and abolished the constrictor effect of PAF on the coronary vessels. These data suggest that PAF might play a detrimental role on the coronary circulation and cardiac function, primarily through thromboxane A₂ mediated mechanism.     

Simultaneous Imaging of CBF Change and BOLD with Saturation-Recovery-T1 Method

A neuroimaging technique based on the saturation-recovery (SR)-T₁ MRI method was applied for simultaneously imaging blood oxygenation level dependence (BOLD) contrast and cerebral blood flow change (ΔCBF), which is determined by CBF-sensitive T₁ relaxation rate change (ΔR₁ ^CBF). This technique was validated by quantitatively examining the relationships among ΔR₁ ^CBF, ΔCBF, BOLD and relative CBF change (rCBF), which was simultaneously measured by laser Doppler flowmetry under global ischemia and hypercapnia conditions, respectively, in the rat brain. It was found that during ischemia, BOLD decreased 23.1±2.8% in the cortical area; ΔR₁ ^CBF decreased 0.020±0.004s^-1 corresponding to a ΔCBF decrease of 1.07±0.24 ml/g/min and 89.5±1.8% CBF reduction (n=5), resulting in a baseline CBF value (=1.18 ml/g/min) consistent with the literature reports. The CBF change quantification based on temperature corrected ΔR₁ ^CBF had a better accuracy than apparent R₁ change (ΔR₁ ^app); nevertheless, ΔR₁ ^app without temperature correction still provides a good approximation for quantifying CBF change since perfusion dominates the evolution of the longitudinal relaxation rate (R₁ ^app). In contrast to the excellent consistency between ΔCBF and rCBF measured during and after ischemia, the BOLD change during the post-ischemia period was temporally disassociated with ΔCBF, indicating distinct CBF and BOLD responses. Similar results were also observed for the hypercapnia study. The overall results demonstrate that the SR-T₁ MRI method is effective for noninvasive and quantitative imaging of both ΔCBF and BOLD associated with physiological and/or pathological changes.     

Prostacyclin inhibits 5-hydroxytryptamine release but stimulates thromboxane synthesis during cardiopulmonary bypass

The antiaggregating agent prostacyclin (PGI₂) was infused into ten dogs during cardiopulmonary bypass (CPB) to minimize thrombocytopenia and platelet dysfunction. The animals were anesthetized, placed on mechanical ventilation and underwent thoracotomy. After heparinization with 300 u/kg, animals were assigned to control (n=5) or PGI₂ treated groups (n=5). Thoracotomy and then CPB decreased platelet numbers to below 30, 000/mm³ (p < 0.05) and fibrinogen to less than 150 mg/dl (p < 0.05). PGI₂ at 100 ng/kg·min was infused for the 2 h period of CPB. PGI₂ infusion did not prevent these changes, but did prevent platelet serotonin release. In the control group after CPB, platelet serotonin fell from the baseline value of 1.11 μg/10⁹ to 0.35 μg/10⁹ platelets (p < 0.05). In contrast, PGI₂ treatment resulted in a serotonin increase to 2.27 μg/10⁹ platelets (p < 0.05). Thromboxane B₂ concentrations of platelets and plasma rose during CPB (p < 0.05). Surprisingly, PGI₂ infusion accentuated this rise in platelet and plasma thromboxane B₂ (p < 0.05). These data indicate that during CPB, an infusion of PGI₂: 1) does not prevent thrombocytopenia; 2) increases platelet serotonin uptake despite, 3) an associated rise in platelet and plasma thromboxane B₂.     

Decreased Soluble Adenylyl Cyclase Activity in Cystic Fibrosis Is Related to Defective Apical Bicarbonate Exchange and Affects Ciliary Beat Frequency Regulation

Human airway cilia contain soluble adenylyl cyclase (sAC) that produces cAMP upon HCO₃⁻/CO₂ stimulation to increase ciliary beat frequency (CBF). Because apical HCO₃⁻ exchange depends on cystic fibrosis transmembrane conductance regulator (CFTR), malfunctioning CFTR might impair sAC-mediated CBF regulation in cells from patients with cystic fibrosis (CF). By Western blot, sAC isoforms are equally expressed in normal and CF airway epithelial cells, but CBF decreased more in CF than normal cells upon increased apical HCO₃⁻/CO₂ exposure in part because of greater intracellular acidification from unbalanced CO₂ influx (estimated by 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence). Importantly, ciliated cell-specific cAMP production (estimated by FRET fluorescence ratio changes of tagged cAMP-dependent protein kinase (PKA) subunits expressed under a ciliated cell-specific promoter) in response to increased apical HCO₃⁻/CO₂ perfusion was higher in normal compared with CF cells. Inhibition of bicarbonate influx via CFTR (CFTR_inh172) and inhibition of sAC (KH7) and PKA activation (H89) led to larger CBF declines in normal cells, now comparable with changes seen in CF cells. These inhibitors also reduced FRET changes in normal cells to the level of CF cells with the expected exception of H89, which does not prevent dissociation of the fluorescently tagged PKA subunits. Basolateral permeabilization and subsequent perfusion with HCO₃⁻/CO₂ rescued CBF and FRET changes in CF cells to the level of normal cells. These results suggest that CBF regulation by sAC-produced cAMP could be impaired in CF, thereby possibly contributing to mucociliary dysfunction in this disease, at least during disease exacerbations when airway acidification is common.     

Pulmonary vascular response to thrombin: Effects of thromboxane synthetase inhibition with OKY-046 and OKY-1581

We examined the effects of thromboxane synthetase inhibition with OKY-1581 and OKY-046 on pulmonary hemodynamics and lung fluid balance after thrombin-induced intravascular coagulation. Studies were made in anesthetized sheep prepared with lyng lymph fistulas. Pulmonary intravascular coagulation was induced by i.v. infusion of α-thrombin over a 15 min period. Thrombin infusion in control sheep resulted in immediate increases in pulmonary artery pressure (P ) and pulmonary vascular resistance (PVR), which associated with rapid 3-fold increase in pulmonary lymph flow (Q̇lym) and a delayed increase in lymph-to-plasma protein concentration (L/P) ratio, indicating an increase in the pulmonary microvascular permeability to proteins. Thrombin-induced intravascular coagulation alos increased arterial thromboxane B₂ (a metabolite of thromboxane A₂) and 6-keto-PGF_1α concentrations (a metabolite of prostacyclin). Both OKY-1581 and OKY-046 prevented thromboxane B₂ and 6-keto-PGF_1α generation. The initial increments in P and PVR were attenuated in both treated groups. The increases in Q̇lym were gradual in the treated groups but attained the same levels as in control group. However, the increases in Q̇lym were associated with decreases in L/P ratio. In both treated groups, the leukocyte count decreased after thrombin infusion but then increased steadily above the baseline value, whereas the leukocyte count remained depressed in the control group after thrombin. These studies indicate that a part of the initial pulmonary vasoconstrictor response to thrombin-induced intravascular coagulation is mediated by thromboxane generation. In addition, thromboxane may also contribute to the increase in lung vascular permeability to proteins that occurs after intravascular coagulation and this effect may be mediated by a thromboxane-neutrophil interaction.     

Predicting Recovery of Voluntary Upper Extremity Movement in Subacute Stroke Patients with Severe Upper Extremity Paresis

DesignProspective cohort study.Methods140 (out of 590) stroke patients with severe UE paresis completed all assessments. Voluntary UE movement was assessed using the UE subscale of the Stroke Rehabilitation Assessment of Movement (STREAM-UE). Two outcome measures, STREAM-UE scores at discharge (DC_STREAM-UE) and changes between admission and discharge (Δ_STREAM-UE), were investigated to represent the final states and improvement of the recovery of voluntary UE movement. Stepwise regression analyses were used to investigate 19 clinical variables and to find the best predictive models of the two outcome measures.ResultsThe participants showed wide variation in both DC_STREAM-UE and Δ_STREAM-UE. 3.6% of the participants almost fully recovered at discharge (DC_STREAM-UE > 15). A large improvement (Δ_STREAM-UE >= 10) occurred in 16.4% of the participants, while 32.9% of the participants did not have any improvement. The four predictors for the DC_STREAM-UE (R² = 35.0%) were ‘baseline STREAM-UE score’, ‘hemorrhagic stroke’, ‘baseline National Institutes of Health Stroke Scale (NIHSS) score’, and ‘cortical lesion excluding primary motor cortex’. The three predictors for the Δ_STREAM-UE (R² = 22.0%) were ‘hemorrhagic stroke’, ‘baseline NIHSS score’, and ‘cortical lesion excluding primary motor cortex’.ConclusionsRecovery of voluntary UE movement varied widely in patients with severe UE paresis after stroke. The predictive power of clinical variables was poor. Both results indicate the complex nature of voluntary UE movement recovery in patients with severe UE paresis after stroke.     

Platelet Activation after Presyncope by Lower Body Negative Pressure in Humans

Central hypovolemia elevates hemostatic activity which is essential for preventing exsanguination after trauma, but platelet activation to central hypovolemia has not been described. We hypothesized that central hypovolemia induced by lower body negative pressure (LBNP) activates platelets. Eight healthy subjects were exposed to progressive central hypovolemia by LBNP until presyncope. At baseline and 5 min after presyncope, hemostatic activity of venous blood was evaluated by flow cytometry, thrombelastography, and plasma markers of coagulation and fibrinolysis. Cell counts were also determined. Flow cytometry revealed that LBNP increased mean fluorescence intensity of PAC-1 by 1959±455 units (P<0.001) and percent of fluorescence-positive platelets by 27±18%-points (P = 0.013). Thrombelastography demonstrated that coagulation was accelerated (R-time decreased by 0.8±0.4 min (P = 0.001)) and that clot lysis increased (LY₆₀ by 6.0±5.8%-points (P = 0.034)). Plasma coagulation factor VIII and von Willebrand factor ristocetin cofactor activity increased (P = 0.011 and P = 0.024, respectively), demonstrating increased coagulation activity, while von Willebrand factor antigen was unchanged. Plasma protein C activity and tissue-type plasminogen activator increased (P = 0.007 and P = 0.017, respectively), and D-dimer increased by 0.03±0.02 mg l⁻¹ (P = 0.031), demonstrating increased fibrinolytic activity. Plasma prothrombin time and activated partial thromboplastin time were unchanged. Platelet count increased by 15±13% (P = 0.014) and red blood cells by 9±4% (P = 0.002). In humans, LBNP-induced presyncope activates platelets, as evidenced by increased exposure of active glycoprotein IIb/IIIa, accelerates coagulation. LBNP activates fibrinolysis, similar to hemorrhage, but does not alter coagulation screening tests, such as prothrombin time and activated partial thromboplastin time. LBNP results in increased platelet counts, but also in hemoconcentration.     

Combined Treatment of Xenon and Hypothermia in Newborn Rats - Additive or Synergistic Effect?

BackgroundBreathing the inert gas Xenon (Xe) enhances hypothermic (HT) neuroprotection after hypoxia-ischemia (HI) in small and large newborn animal models. The underlying mechanism of the enhancement is not yet fully understood, but the combined effect of Xe and HT could either be synergistic (larger than the two effects added) or simply additive. A previously published study, using unilateral carotid ligation followed by hypoxia in seven day old (P7) rats, showed that the combination of mild HT (35°C) and low Xe concentration (20%), both not being neuroprotective alone, had a synergistic effect and was neuroprotective when both were started with a 4 h delay after a moderate HI insult. To examine whether another laboratory could confirm this finding, we repeated key aspects of the study.Design/MethodsAfter the HI-insult 120 pups were exposed to different post-insult treatments: three temperatures (normothermia (NT) NT_37°C, HT_35°C, HT_32°C) or Xe concentrations (0%, 20% or 50%) starting either immediately or with a 4 h delay. To assess the synergistic potency of Xe-HT, a second set (n = 101) of P7 pups were exposed to either HT_35°C+Xe_0%, NT+Xe_20% or a combination of HT_35°C+Xe_20% starting with a 4 h delay after the insult. Brain damage was analyzed using relative hemispheric (ligated side/unligated side) brain tissue area loss after seven day survival.ResultsImmediate HT_32°C (p = 0.042), but not HT_35°C significantly reduced brain injury compared to NT_37°C. As previously shown, adding immediate Xe_50% to HT_32°C increased protection. Neither 4 h-delayed Xe_20%, nor Xe_50% at 37°C significantly reduced brain injury (p>0.050). In addition, neither 4 h-delayed HT_35°C alone, nor HT_35°C+Xe_20% reduced brain injury. We found no synergistic effect of the combined treatments in this experimental model.ConclusionsCombining two treatments that individually were ineffective (delayed HT_35°C and delayed Xe_20%) did not exert neuroprotection when combined, and therefore did not show a synergistic treatment effect.     

The Effect of Adding CO2 to Hypoxic Inspired Gas on Cerebral Blood Flow Velocity and Breathing during Incremental Exercise

Hypoxia increases the ventilatory response to exercise, which leads to hyperventilation-induced hypocapnia and subsequent reduction in cerebral blood flow (CBF). We studied the effects of adding CO₂ to a hypoxic inspired gas on CBF during heavy exercise in an altitude naïve population. We hypothesized that augmented inspired CO₂ and hypoxia would exert synergistic effects on increasing CBF during exercise, which would improve exercise capacity compared to hypocapnic hypoxia. We also examined the responsiveness of CO₂ and O₂ chemoreception on the regulation ventilation (E) during incremental exercise. We measured middle cerebral artery velocity (MCAv; index of CBF), E, end-tidal PCO₂, respiratory compensation threshold (RC) and ventilatory response to exercise (E slope) in ten healthy men during incremental cycling to exhaustion in normoxia and hypoxia (FIO₂ = 0.10) with and without augmenting the fraction of inspired CO₂ (FICO₂). During exercise in normoxia, augmenting FICO₂ elevated MCAv throughout exercise and lowered both RC onset andE slope below RC (P<0.05). In hypoxia, MCAv and E slope below RC during exercise were elevated, while the onset of RC occurred at lower exercise intensity (P<0.05). Augmenting FICO₂ in hypoxia increased E at RC (P<0.05) but no difference was observed in RC onset, MCAv, or E slope below RC (P>0.05). The E slope above RC was unchanged with either hypoxia or augmented FICO₂ (P>0.05). We found augmenting FICO₂ increased CBF during sub-maximal exercise in normoxia, but not in hypoxia, indicating that the ‘normal’ cerebrovascular response to hypercapnia is blunted during exercise in hypoxia, possibly due to an exhaustion of cerebral vasodilatory reserve. This finding may explain the lack of improvement of exercise capacity in hypoxia with augmented CO₂. Our data further indicate that, during exercise below RC, chemoreception is responsive, while above RC the ventilatory response to CO₂ is blunted.     

Hypersensitivity to thromboxane receptor mediated cerebral vasomotion and CBF oscillations during acute NO-deficiency in rats

Background

Low frequency (4–12 cpm) spontaneous fluctuations of the cerebrovascular tone (vasomotion) and oscillations of the cerebral blood flow (CBF) have been reported in diseases associated with endothelial dysfunction. Since endothelium-derived nitric oxide (NO) suppresses constitutively the release and vascular effects of thromboxane A₂ (TXA₂), NO-deficiency is often associated with activation of thromboxane receptors (TP). In the present study we hypothesized that in the absence of NO, overactivation of the TP-receptor mediated cerebrovascular signaling pathway contributes to the development of vasomotion and CBF oscillations.

Methodology/Principal Findings

Effects of pharmacological modulation of TP-receptor activation and its downstream signaling pathway have been investigated on CBF oscillations (measured by laser-Doppler flowmetry in anesthetized rats) and vasomotion (measured by isometric tension recording in isolated rat middle cerebral arteries, MCAs) both under physiological conditions and after acute inhibition of NO synthesis. Administration of the TP-receptor agonist U-46619 (1 µg/kg iv.) to control animals failed to induce any changes of the systemic or cerebral circulatory parameters. Inhibition of the NO synthesis by nitro-L-arginine methyl esther (L-NAME, 100 mg/kg iv.) resulted in increased mean arterial blood pressure and a decreased CBF accompanied by appearance of CBF-oscillations with a dominant frequency of 148±2 mHz. U-46619 significantly augmented the CBF-oscillations induced by L-NAME while inhibition of endogenous TXA₂ synthesis by ozagrel (10 mg/kg iv.) attenuated it. In isolated MCAs U-46619 in a concentration of 100 nM, which induced weak and stable contraction under physiological conditions, evoked sustained vasomotion in the absence of NO, which effect could be completely reversed by inhibition of Rho-kinase by 10 µM Y-27632.

Conclusion/Significance

These results suggest that hypersensitivity of the TP-receptor – Rho-kinase signaling pathway contributes to the development of low frequency cerebral vasomotion which may propagate to vasospasm in pathophysiological states associated with NO-deficiency.     

Thromboxane induced red blood cell lysis

The two thromboxane A₂ mimetics, carbocyclic thromboxane A₂ (CTA₂) and U-46619 (9,11-methanoepoxy PGH₂) at concentrations of 400 ng/ml significantly enhanced the release of hemoglobin from both feline and human erythrocyte suspensions. This effect was significantly attenuated by the thromboxane receptor antagonist BM-13,505 indicating that the membrane leakiness is in some way receptor mediated. The effects also appear to be concentration-dependent over the range of 100–400 ng/ml. The membrane labilizing effect of thromboxane analogs is not due to a non-specific eicosanoid effect since iloprost, the stable prostacyclin analog, actually stabilized erythrocyte membranes. Moreover, synthetic thromboxane A₂ exerted similar effects to that of the two TxA₂-mimetics. This membrane labilizing action of thromboxanes may be important in propagating the other pathophysiologic effects of thromboxane A₂ in cardiovascular disease states.     

Testing the “redirection hypothesis” of prostaglandin metabolism in the kidney

Furosemide increases the synthesis of two major renal eicosanoids, prostacylin (PGI₂) and thromboxane A₂ (TXA₂), by stimulating the release of arachidonic acid which in turn is metabolized to PGG₂/PGH₂, then to PGI₂ and TXA₂. PGI₂ may mediate, in part, the early increment in plasma renin activity (PRA) after furosemide. We hypothesized that thromboxane synthetase inhibition should direct prostaglandin endoperoxide metabolism toward PGI₂, thereby enhancing the effects of furosemide on renin release. Furosemide (2.0 mg.kg⁻¹ i.v.) was injected into Sprague-Dawley rats pretreated either with vehicle or with U-63, 557A (a thromboxane synthetase inhibitor, 2 mg/kg⁻¹ followed by 2 mg/kg⁻¹.hr⁻¹). Urinary 6ketoPGF₁ α and thromboxane B₂ (TXB₂), reflecting renal synthesis of PGI₂ and TXA₂, as well as PRA and serum TXB₂, were measured. Serum TXB₂ was reduced by 96% after U-63, 557A. U-63, 557A did not affect the basal PRA. Furosemide increased PRA in both vehicle and U63, 557A treated rats. However, the PRA-increment at 10, 20 and 40 min following furosemide administration was greater in U-63, 557A-treated rats than in vehicle-treated rats and urine 6ketoPGF₁ α excretion rates were increased. These effects of thromboxane synthesis inhibition are consistent with a redirection of renal PG synthesis toward PGI₂ and further suggest that such redirection can be physiologically relevant.     

The effect of acute hypoxia on prostaglandin release in perfused human fetal placenta

The release of prostaglandin E₂ and F_2α, thromboxane B₂ and 6-keto-prostaglandin F_1α was measured in isolated human placental cotyledons perfused under high- and low-oxygen conditions. Also the effect of reoxygenation on prostaglandin production was studied. During the high-oxygen period, prostaglandin E₂ accounted for 44 % and 6-keto-prostaglandin F_1α for 28 % of all prostaglandin release, and the rank order of prostaglandin release was E₂ > 6-keto-prostaglandin F_1α > thromboxane B₂ > prostaglandin F_2α. Hypoxia had no significant effect on quantitative prostaglandin release, but the ration of prostaglandin E₂ to prostaglandin F_2α was significantly increased. After the hypoxic period during reoxygenation the release of 6-keto-prostaglandin F_1α was significantly decreased, as was the ratio of 6-keto-prostaglandin F_1α to thromboxane B₂. Also the ratio of the vasodilating prostaglandins (E₂, 6-keto-prostaglandin F_1α) to the vasocontricting prostaglandins (thromboxane B₂, prostaglandin F_2α) was decreased during reoxygenation period. With the constant flow rate, the perfusion pressure increased during hypoxia in six and was unchanged in three preparation. The results indicate that changes in the tissue oxygenation in the placenta affect prostaglandin release in the fetal placental circulation. This may also have circulatory consequences.     

N-3 Fatty Acid Rich Triglyceride Emulsions Are Neuroprotective after Cerebral Hypoxic-Ischemic Injury in Neonatal Mice

We questioned if acute administration of n-3 fatty acids (FA) carried in n-3 rich triglyceride (TG) emulsions provides neuroprotection in neonatal mice subjected to hypoxic-ischemic (H/I) brain injury. We examined specificity of FA, optimal doses, and therapeutic windows for neuroprotection after H/I. H/I insult was induced in C57BL/6J 10-day-old mice by right carotid artery ligation followed by exposure to 8% O₂ for 15 minutes at 37°C. Intraperitoneal injection with n-3-rich TG emulsions, n-6 rich TG emulsions or saline for control was administered at different time points before and/or after H/I. In separate experiments, dose responses were determined with TG containing only docosahexaenoic acid (Tri-DHA) or eicosapentaenoic acid (Tri-EPA) with a range of 0.1–0.375 g n-3 TG/kg, administered immediately after H/I insult. Infarct volume and cerebral blood flow (CBF) were measured. Treatment with n-3 TG emulsions both before- and after- H/I significantly reduced total infarct volume by a mean of 43% when administered 90 min prior to H/I and by 47% when administered immediately after H/I. In post-H/I experiments Tri-DHA, but not Tri-EPA exhibited neuroprotective effects with both low and high doses (p<0.05). Moreover, delayed post-H/I treatment with Tri-DHA significantly decreased total infarct volume by a mean of 51% when administered at 0 hr, by 46% at 1 hr, and by 51% at 2 hr after H/I insult. No protective effect occurred with Tri-DHA injection at 4 hr after H/I. There were no n-3 TG related differences in CBF. A significant reduction in brain tissue death was maintained after Tri-DHA injection at 8 wk after the initial brain injury. Thus, n-3 TG, specifically containing DHA, is protective against H/I induced brain infarction when administered up to 2 hr after H/I injury. Acute administration of TG-rich DHA may prove effective for treatment of stroke in humans.     

Cerebral cortical blood flow in rabbits during parabolic flights (hypergravity and microgravity)

We studied the effect of gravity on cerebral cortical blood flow (CBF), mean arterial blood pressure () and heart rate in six rabbits exposed to parabolic flights. The CBF was obtained using a laser-Doppler probe fixed on to a cranial window. Before weightlessness, the animals were exposed to chest-to-back directed acceleration (1.8–2.0 g). The CBF values were expressed as a percentage of CBF_o (mean CBF during 60 s before the 1st parabola). Propranolol (1 mg · kg⁻¹ IV) was given after the 11th parabola and pentobarbital (12–15 mg · kg⁻¹ IV) after the 16th parabola. Before the administration of the drugs, CBF increased (P < 0.01) during hypergravity [i.e. maximal CBF 151 (SD 64)% CBF_o. Simultaneously increased [maximal , 119 (SD 11) mmHg (P < 0.01)]. At the onset of weightlessness, CBF and reached maximal values [194 (SD 96)% CBF_o (P < 0.01) and 127 (SD 19) mmHg, (P < 0.01) respectively]. The microgravity-induced increase in CBF was transient since CBF returned to its baseline value after 8 (SD 2) s of microgravity. After propranolol administration, CBF was not statistically different during hypergravity but an elevation of CBF was still observed in weightlessness. The increases in CBF and also persisted during weightlessness after pentobarbital administration. These data would indicate that CBF of nonanesthetized rabbits increases during the first seconds of weightlessness and demonstrate the involvement of rapid active regulatory mechanisms since CBF returned to control values within 8 (SD 2) s. We concluded that this elevation in blood flow was not related to stress because it persisted after the administration of propranolol and pentobarbital. Accepted: 6 November 1997     

Calcium and the regulation of mammalian ciliary beating

Summary This report summarizes our recent work on the role of intracellular Ca²⁺ ([Ca²⁺]_i) in regulating mammalian ciliary beat frequency (CBF). CBF from a single ovine cilium and [Ca²⁺]_i from the same cell were measured by digital video phase contrast microscopy and fura-2 ratiometric imaging video microscopy, respectively. Cells were stimulated with two exposures to 10 M acetylcholine (ACh). CBF was recorded during the first and [Ca²⁺]_i during the second stimulation. ACh increased [Ca²⁺]_i and CBF transiently with indistinguishable kinetics and, early in culture, even induced [Ca²⁺]_i oscillations and ciliary frequency modulations with the same peak-to-peak time interval. Cells treated with 1 M thapsigargin, an inhibitor of the endoplasmic-reticulum Ca²⁺-ATPase, showed transient [Ca²⁺]_i and CBF increases, again with similar kinetics, which often remained at an elevated plateau. Application of ACh to cells pretreated with thapsigargin produced decreases in both [Ca²⁺]_i and CBF. Finally, changing extracellular Ca²⁺-concentrations induced corresponding changes in [Ca²⁺]_i that were associated with kinetically similar CBF changes. These data strongly suggested that [Ca²⁺]_i is a critical signal to regulate CBF in mammalian tracheal epithelial cells. In an initial effort to provide constraints on the number and type of reactions that link changes in [Ca²⁺]_i to changes in CBF, simultaneous recordings of both signals from a single cell were analyzed. Such recordings provided higher resolution of the kinetic responses of CBF and [Ca²⁺]_i to ACh as well as they allowed direct assessment of the coupling between [Ca²⁺]_i and CBF. Simultaneous measurements revealed that [Ca²⁺]_i and CBF were perfectly correlated within the CBF measurement time resolution, except for the period of the fastest changes in both signals during the initial ACh exposure. There, changes in CBF lagged the changes in [Ca²⁺]_i by 1–3 ciliary beat cycles (ca. 150–450 ms).     

Electron capture gas chromatographic detection of thromboxane B2

An electron capture gas chromatographic method is described for the detection of thromboxane B₂. Thromboxane B₂ is esterified with diazomethane, followed by treatment with pentafluorobenzylhydroxylamine hydrochloride and silylation with BSA. In pyridine, the free aldehyde form of the acetal ring is favored allowing rapid formation of a novel thromboxane B₂ pentafluorobenzyloxime. The method has been applied to detect thromboxane B₂ formation during aggregation of washed platelets. It must be emphasized that by ordinary analytical standards, the derivatization reproducibility from 50–375 nanograms is poor (±11% – ±42%); however, the improved selectivity of the method and its ability to detect nanogram levels of thromboxane B₂ make it a useful complement to commonly employed bioassay techniques.     

Cardiac and coronary consequences of intracoronary platelet activating factor infusion in the domestic pig

In previous studies we have shown that platelet-activating factor (PAF) is a potent vasoactive substance with deleterious effects on coronary blood flow (CBF) and myocardial performance. The present study further investigates the effects of PAF during its sustained intracoronary infusion in the blood-perfused domestic pig (n = 16). PAF infusion (1-9 nmol/min) produced triphasic changes in CBF (n = 7): an initial brief phase of coronary dilation (14 +/- 2% above baseline), followed by severe reduction in CBF due to increase in coronary vascular resistance and a third phase of escape that was characterized by return of CBF towards baseline in spite of continuing PAF infusion. In 9 remaining pigs PAF infusion had a biphasic response: the first phase of coronary dilation rapidly turned into severe coronary constriction accompanied by severe systemic hypotension and death within a few min. PAF infusion caused a profound rise in systemic arterial and coronary venous thromboxane B2 levels, while 6-keto-PGF1 alpha and leukotriene C4-immunoreactivity levels were not changed. Indomethacin completely blocked the rise in thromboxane level during PAF infusion and abolished the constrictor effect of PAF on the coronary vessels. These data suggest that PAF might play a detrimental role on the coronary circulation and cardiac function, primarily through thromboxane A2 mediated mechanism.     

Coronary vasoconstrictor extracted from blood plasma stimulates platelet lipoxidase activity

The effects of a coronary vasoconstrictor, obtained from human blood plasma, on aggregation and arachidonate metabolism by human platelets was determined. At low concentrations, the vasoactive factor stimulated formation of prostaglandins, thromboxane B₂, and 12-L-hydroxyeicosatetraenoic acid in both intact platelets and in platelet microsomal enzyme preparations. As factor concentration was increased, thromboxane B₂ formation decreased, but production of the other products continued to rise. Low concentrations of factor initiated platelet aggregation, whereas high concentrations prevented arachidonate-induced aggregation. Low levels of factor could induce aggregation via stimulation of thromboxane A₂ production. Increases in formation of 12-hydroperoxyeicosatetraenoic acid at high factor concentrations could inhibit formation of thromboxane A₂ and thus prevent aggregation.