Recombinant human activated factor VII is thrombogenic in a rabbit model of cyclic flow reduction and does not reduce intra-abdominal bleeding

Recombinant human activated factor VII (rHuFVIIa) can reduce bleeding but may be associated with arterial thrombosis. We hypothesized that rHuFVIIa would increase the occurrence of cyclic flow reductions (CFR) and reduce intra-abdominal bleeding in an experimental model. An adapted Folts' model of carotid artery lesion and stenosis was used. Twenty four rabbits were randomized to receive rHuFVIIa (group F) or placebo (group P) in a double-blind fashion. A standardized injury to the common carotid artery resulted in CFR and/or thrombosis. Hematological values, coagulation and thromboelastographic (TEG) variables were compared. Intra-abdominal bleeding was evaluated by measuring blood loss from standardized hepatosplenic lesions. The median number (range) of spontaneous CFR [group P: 6 (0-15); group F: 8 (0-16)] was comparable between groups. The number of induced CFR (by "shaking" of the artery) needed to avert thrombosis (group F: 2; group P: 0; p < 0.05) and the incidence of complete carotid artery thrombosis (group F: 3; group P: 0; p < 0.05) were higher in group F. Intra-abdominal bleeding was similar in both groups. TEG analysis demonstrated a hypercoagulable state in both groups but the magnitude of the change was statistically more important in group F. rHuFVIIa increases thrombosis in a rabbit model of carotid artery injury. The bleeding from hepatic and splenic lesions is not reduced by administration of rHuFVIIa despite a hypercoagulable state confirmed by standard TEG analysis.     

Microangiopathy, the vascular basement membrane and Alzheimer''s disease: a review

The present review focuses on the vascular basement membrane (VBM) and its relationship to the lesions of Alzheimer's disease (AD). Examination of the fine structure of the microvasculature reveals AD-associated VBM alterations, which include both thickening and vacuolization. Immunocytochemistry confirms that all three intrinsic VBM components [collagen type IV, laminin, and heparan sulfate proteoglycan (HSPG)] outline the capillary bed, which is pathologically altered in AD patients (microangiopathy). Ultrastructural analyses of AD tissue samples demonstrate that HSPG's normal staining pattern is disrupted on the endothelial surface of the VBM in brain regions affected by Alzheimer lesions. Similarly altered VBM is reported to occur in the kidney of patients with diabetes mellitus, where it is associated with a leakage of protein. All three VBM components immunolabel capillaries, amyloid and plaque-associated glial processes, suggesting a link between microangiopathy and senile plaque formation. In addition, the consistent colocalization of HSPG with several forms of amyloid implies an involvement in amyloidogenesis. Finally, the neurotrophic effects of beta-amyloid, combined with neurite-promoting effects of laminin and HSPG, could create a strong focus for an aberrant sprouting response. Such a response is postulated to result in plaque-associated degenerating neurites. Thus, VBM components could serve as a nidus for plaque formation, playing a role in the development of neuritic as well as amyloidotic elements.     

高脂高糖家兔脑梗死模型基底膜损伤机制的探讨

目的 探讨脑缺血后微血管内皮细胞基底膜的损伤机制.方法 24只新西兰家兔采用随机数字表法分为非高脂高糖脑梗死模型对照组(简称空白对照组),高脂高糖脑梗死模型对照组(简称模型对照组),每组12只.空白对照组给予普通饲料,模型对照组给予高脂高糖饲料,均制作成脑梗死模型.监测造模前后血浆中层粘连蛋白(LN)的表达,并观察腩组织中LN表达情况.结果模型对照组高脂高糖模型制作后,家兔血脂血糖增高,与造模前比较差异有统计学意义(P<0.05).脑梗死模型制作后两组血浆中LN表达减少,与造模前比较差异有统计学意义(P<0.05).病理观察结果显示LN主要表达在微血管基质,阳性反应为黄色到棕褐色.结论 兔脑梗死模型中早期即有微血管内皮细胞基底膜的损伤,表现在细胞外摹质中LN的降低.在脑缺血早期积极干预患者血糖血脂水平及联合运用脑保护剂,MMPs抑制剂将有效改善梗死程度.     

Prostacyclin biosynthesis in vascular endothelium is not inhibited by cyclic AMP. Studies with 3-isobutyl-1-methylxanthine and forskolin

We have previously reported ($\text{Proc. Natl. Acad. Sci.}$ 79, 495–499, 1982) that the cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine (IBMX), stimulates cyclic AMP accumulation and inhibits prostacyclin (PGI₂) production in primary monolayer cultures of human umbilical vein endothelium. The present study was carried out to determine whether these effects are causally related. Incubation of endothelial monolayers with the diterpene, forskolin, increased the intracellular concentration of cyclic AMP by 10-fold. Despite this marked increase in cyclic AMP, neither baseline production of PGI₂ nor release in response to stimulation by thrombin or the divalent cation ionophore, A23187, was affected. Both forskolin and isoproterenol were found to potentiate the effect of IBMX on cyclic AMP accumulation without causing further inhibition of PGI₂ biosynthesis. Inhibition of cyclic nucleotide phosphodiesterase activity with 2,6-bis-(diethanolamino)-4-piperidinopyrimido-[5,4-d]pyrimidine increased cyclic AMP levels to the same extent as IBMX; however, this agent had no effect on PGI₂ biosynthesis. These findings demonstrate that increases in the intracellular concentration of cyclic AMP have no short-term effects on PGI₂ biosynthesis in vascular endothelium and suggest that inhibition of PGI₂ production by IBMX is the result of some other, cyclic AMP-independent action of the drug.     

An ultrastructural study of the vascular and muscular basement membrane in Duchenne-type dystrophy

The ultrastructural distribution of basement membrane around the capillaries as well as muscle cells of boys with Duchenne-type muscular dystrophy was determined. In dystrophic muscles, a diffuse thickness of vascular as well as muscular basement membrane was observed. Lamina densa lost its regular ribbon-like appearance and was split into several thin layers. After staining with tannic acid (TA), a densely stained meshwork was present on the muscle cell surface as well as in the extra-cellular space. Hyaluronidase treatment removed TA-stained deposits, indicating that hyaluronic acid is a major component. Enzyme resistant structures, presumably fibrous long-spacing collagen fibrils, have been found in dystrophic muscles. Based on the results of the study, faulty structure of the basement membrane in dystrophic muscle is suggested.     

Regional sensitivity to neuroinflammation: in vivo and in vitro studies

Background: Neuroinflammation is involved in several acute‐onset neuropathologies such as meningitis, encephalitis, stroke, and traumatic brain injury as well as in neurodegenerative diseases. All of these patholologies are associated with cognitive deficits. Using a model of pure neuroinflammation (intracisternal injection of endotoxin in mice), we tested the hypothesis that brain regions involved in cognition are the most vulnerable to inflammatory insults, and this vulnerability is an inherent property of neocortical neurons. Methods: Mice (n = 10/group) injected with endotoxin (LPS) or saline in the cisterna magna underwent neurobehavioral and cognitive testing followed by quantitative autoradiographic assessment of regional neuroinflammation with [3H]PK11195, an established marker of microgliosis. In parallel, cocultures of cortical and striatal neurons taken from embryonic day 19 rat embryos or postnatal day 1 mice expressing green fluorescent protein were exposed for 24 h to the proinflammatory cytokine TNFalpha, glutamate, or a combination of the two agents. Results: LPS‐treated mice exhibited significant deficits in memory and significant increases in specific PK11195 binding in cortical and hippocampal regions, but not in striatum. Cultured neurons of cortical origin showed significantly lower survival rate relative to striatal neurons in response to TNFalpha, glutamate, or a combination of the two agents. Furthermore, TNFalpha exerted neuroprotective rather than neurotoxic effects in the striatal but not in the cortical neurons. Conclusions: These results suggest that the cortex is inherently more sensitive than the striatum to the deleterious effects of neuroinflammation, and may offer an explanation for the preponderance of cognitive deficits in neuropathologies with a neuroinflammatory component. Synapse, 2011. © 2010 Wiley‐Liss, Inc.     

The presence of plasma proteins facilitates the uptake of 125I-thrombin by the rabbit thoracic aorta endothelium in vitro

Various purified proteins, protein derivatives and two polysaccharides were added individually to a physiological medium in order to effect uptake of 125I-thrombin by the rabbit aorta endothelium. Over a wide range of concentration (0.004-40 mg/ml), the presence of either purified rabbit or bovine albumin during thrombin uptake encouraged an increase (70-110%) in 125I-thrombin binding by the endothelium and subendothelium compared to uptake by aorta segments in the absence of added protein. Pretreatment of aorta segments with albumin before incubation with 125I-thrombin in the absence of albumin did not encourage thrombin uptake to the same extent as having 125I-thrombin and albumin together. Purified human transferrin, rabbit IgG, chicken ovalbumin or denatured bovine casein could replace albumin to produce a similar enhancement of thrombin uptake. Replacing active concentrations of albumin by either reduced-carboxymethylated albumin, defatted albumin, plasmin-treated or thermolysin-treated albumin also caused an increase (50-130%) in thrombin binding, whereas replacement by acid-hydrolysed albumin or with polyglutamic acid was either ineffective or even inhibitory. Lysine-modified or arginine-modified albumins caused a small enhancement (14-32%) and no enhancement of thrombin uptake, respectively. Dextran, at low concentration (0.04-0.4 mg/ml) did not influence thrombin uptake, and at higher concentration (4-40 mg/ml) caused a decrease in uptake by both the endothelium and subendothelial layers. Low concentration of dextran sulphate inhibited thrombin uptake to 20-30% of control values. These data express the importance of accompanying protein in the response of the vascular endothelium during binding of thrombin. The possibility that other protein-cell interactions may be similarly influenced by macromolecular solutes is also discussed.     

Distribution of cytochrome oxidase in rat brain: studies with diaminobenzidine histochemistry in vitro and [14C]cyanide tissue labeling in vivo

Studies in experimental animals and post-mortem studies in humans have indicated that the level of the mitochondrial enzyme cytochrome oxidase within brain anatomical pathways is regulated by the long-term functional use of those pathways. To study this relationship, we have measured cytochrome oxidase spectrophotometrically in punch biopsies from different brain regions of rat. We compared these assays against results from the diaminobenzidine histochemical technique. We found a high degree of correlation (r = 0.90) between the density of diaminobenzidine reaction product and enzyme activity. This validates the usefulness of the diaminobenzidine technique for anatomical localization and measurement of this enzyme. To study the feasibility of using radioactive cyanide as an in vivo ligand of cytochrome oxidase, we performed quantitative autoradiographic analysis of rat brains of animals given an intravenous bolus injection of [14C]cyanide. Analysis of the arterial blood curve indicated a complex redistribution of cyanide between red blood cells, plasma, and tissues. Brain labeling reached peak levels at 1 min and then fell despite rising concentrations of free plasma cyanide. Analysis of autoradiographic images revealed good anatomical resolution. The density of labeling in individual structures over time failed to show a strong correlation with cytochrome oxidase activity or diaminobenzidine reaction product.     

Embryogenesis of the avian iris sphincter muscle: in vivo and in vitro studies

The histogenesis of iris sphincter muscle was studied in birds. Chick embryo iris "anlagen", ages from 3 days (st. 18 H.H.) to hatching, were examined. At the 4th day (st. 24 H.H.), nerve fibers were observed in the mesenchyme of the inferotemporal quadrant of the optic cup near the colobomic fissue. Among the mesenchymal cells, there were cells characterized by AChE activity, presence of desmin filaments, exhibiting ACh receptors, and ultrastructurally similar to the presumptive skeletal myoblasts. One day later (st. 27 H.H.), these myoblasts could be cultivated. The formation of myotubes began between 10 and 12 days. From 9 to 14 days, cells left the anterior epithelium of the iris to give rise to the smooth iris muscle; during this evolution some epithelial cells fused with the myotubes taking part in the histogenesis of striated muscle. The possibility of a neurogenic determination for the iris skeletal muscle is discussed.     

Perinatal maturation of the mouse respiratory rhythm-generator: in vivo and in vitro studies

In vivo (plethysmography) and in vitro (en bloc preparations) experiments were performed from embryonic day 16 (E16) to postnatal day 9 (P9) in order to analyse the perinatal maturation of the respiratory rhythm-generator in mice. At E16, delivered foetuses did not ventilate and survive but at E18 they breathed at about 110 cycles/min with respiratory cycles of variable individual duration. From E18 to P0-P2, the respiratory cycles stabilised without changes in the breathing parameters. However, these increased several-fold during the next days. Hypoxia increased breathing frequency from E18-P5 and only significantly affected ventilation from P3 onwards. At E16, in vitro medullary preparations (pons resection) produced rhythmic phrenic bursts at a low frequency (about 5 cycles/min) with variable cycle duration. At E18, their frequency doubled but cycle duration remained variable. After birth, the frequency did not change although cycle duration stabilised. At E18 and P0-P2, the in vitro frequency decreased by around 50% under hypoxia, increased by 40-50% under noradrenaline or substance P and was permanently depressed by the pontine A5 areas. At E16 however, hypoxia had no effects, both noradrenaline and substance P drastically increased the frequency and area A5 inhibition was not expressed at this time. At E18 and P0-P2, electrical stimulation and electrolytic lesion of the rostral ventrolateral medulla affected the in vitro rhythm but failed to induce convincing effects at E16. Thus, a major maturational step in respiratory rhythmogenesis occurs between E16-E18, in agreement with the concept of multiple rhythmogenic mechanisms.     

Estradiol is a protective factor in the adult and aging brain: understanding of mechanisms derived from in vivo and in vitro studies

We have shown that 17β-estradiol exerts profound protective effects against stroke-like ischemic injury in female rats. These effects are evident using physiological levels of estradiol replacement in ovariectomized rats and require hormone treatment prior to the time of injury. The protective actions of estradiol appear to be most prominent in the cerebral cortex, where cell death is not apparent until at least 4 h after the initiation of ischemic injury and where cell death is thought to be apoptotic in nature. Middle-aged rats remain equally responsive to the protective actions of estradiol. The maintenance of responsiveness of the cerebral cortex to the neuroprotective actions of estradiol was unexpected since responsiveness of the hypothalamus to estradiol decreases dramatically by the time animals are middle-aged. We believe that the protective actions of estradiol require the estrogen receptor-α, since estradiol does not protect in estrogen receptor-α knockout mice. We have also implemented a method of culturing cerebral cortical explants to assess the protective effects of estradiol in vitro. This model exhibits remarkable parallelisms with our in vivo model of brain injury. We have found that 17β-estradiol decreases the extent of cell death and that this protective effect requires hormone pretreatment. Finally, 17α-estradiol, which does not interact effectively with the estrogen receptor, does not protect; and addition of ICI 182,780, an estrogen receptor antagonist, blocks the protective actions of estradiol. We have begun to explore the molecular and cellular mechanisms of estradiol-mediated protection. In summary, our findings demonstrate that estradiol exerts powerful protective effects both in vivo and in vitro and suggest that these actions are mediated by estrogen receptors.     

The mechanism of perturbation in monoamine metabolism by L-DOPA therapy: in vivo and in vitro studies

Summary In the cerebrospinal fluid of the patients with Parkinson's disease treated with L-DOPA, L-3-O-methyldopa was the major metabolite of administered L-DOPA. Using a dopaminergic cell model, clonal rat phenochromocytoma PC 12h cells, and by microdialysis of the rat striatum it was proved that L-3-O-methyldopa was taken up into monoamine neurons by transport system specific for aromatic L-amino acids and inhibited transport of L-DOPA and other amino acids competitively. L-3-O-Methyldopa depleted allosteric regulation of the biopterin cofactor on activity of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine synthesis. Depletion of the allostery may perturb the buffer action of endogenous L-DOPA synthesis that stabilizes dopamine level in the brain. By these mechanisms L-3-O-methyldopa may reduce clinical effectiveness of administrated L-DOPA and be involved in wearing-off phenomenon. L-DOPA inhibited the activity of tryptophan hydroxylase and thus serotonin synthesis, which may be related to psychiatric side-effects in the patients under L-DOPA therapy.     

Interactions of amineptine with the neuronal dopamine uptake system: Neurochemicalin vitro andin vivo studies

Summary The effects of amineptine on³H-dopamine uptake and 14C-dopamine release have been studied simultaneously in double labelling test performed on rat striatal synaptosomes.³H-dopamine uptake was completely inhibited at 10M amineptine, a concentration which produced only a weak 14C-DA release (13% of the 14C-radioactivity stored). The IC 50 for the inhibition of³H-DA uptake was not modified by a previous treatment with reserpine whereas the IC 50 of (+) amphetamine and the IC 50 of clomipramine were decreased 9 fold and increased two fold, respectively. In binding studies on rat striatal membranes amineptine displacesin vitro the³H-GBR 12783, bound specifically to a component of the neuronal DA uptake complex. The apparent affinity of amineptine for this binding site was more than 150 times higher than its affinity for the binding site of³H-desipramine on rat cortical membranes. In mice, increasing doses of amineptine injected i. p. reduced in a dose dependent manner the specific retention of radioactivity in the striatum after an i. v. injection of a tracer dose of³H-GBR 12783. These data indicate that amineptine inhibits DA uptake and is virtually devoid of DA releasing effects. It displays a relatively low affinity for the NE uptake system. Its neurochemical profile in the double labelling test clearly differs from that of (+) amphetamine and from that of classical tricyclic anti-depressants.     

Interaction of beta-casomorphins with multiple opioid receptors: in vitro and in vivo studies in the newborn rat brain

beta-Casomorphins (beta-CMs) are peptidic fragments of bovine beta-casein with potent opioid activity. In view of a possible physiological meaning of these milk-derived compounds, we have studied the affinity of some natural beta-CMs and some semisynthetic analogues for mu-, delta- and kappa-brain opioid receptors in newborn and adult rat. Moreover we have investigated whether a chronic treatment with the potent analogue D-Ala2-beta-CM-4-NH2 during the suckling period could affect mu and delta opioid receptor function. Our findings demonstrate that beta-CMs are mu-oriented compounds both in adult and in newborn rat brain. They display the same mu-affinity in newborn as well as in adult animals, however delta and kappa-affinities appear different, probably due to the lower degree of maturation of these two receptors in the first days of life. A prolonged treatment with D-Ala2-beta-CM-4-NH2 during the preweanling period is able to induce a delay in the ontogenetic increase of delta-receptor affinity, whereas it affects neither the affinity nor the density of mu-receptors. This effect could be related to a general action of opioids on cerebral maturative processes; moreover we point out that a beta-CMs analogue, given orally to newborn animals, can induce modifications at central level, suggesting thus the hypothesis that beta-CMs could be a biologically active peptide in the first stages of life.     

Ontogeny of GABAergic neurons in chick brain: studies in vivo and in vitro

The ontogeny of presynaptic elements of GABAergic neurons has been studied in the cerebrum of the chick embryo both in vivo and in vitro. The specific activity of glutamate decarboxylase (GAD) in tissue extracts followed a rising curve and approached a plateau value after 28 days in vivo. One-half of the adult levels of GAD were achieved by day 20. The specific activity of Na+-gamma-aminobutyric acid (GABA) cotransport in membrane vesicles followed a similar pattern and reached maximal levels by 28 days in vivo. One-half of the adult levels of GABA uptake were observed at day 17. The development of these markers was also studied in cultured neurons prepared from the cerebrum of 8-day-old chick embryos. The GAD activity in neuronal extracts increased linearly with time in culture up to 14 days. At this point the specific activity had reached 20% of that observed for the adult cerebrum. The specific activity of GABA uptake by intact neurons followed a pattern similar to that for GAD from days 2 to 9 in culture. Both activities increased 4-5-fold during this period, but the level of GABA transport declined thereafter. In order to compare GABA uptake values for cultured cells with those for embryonic and adult brain, membrane vesicles were prepared from cultures. At the maximal level (9-10 days in culture) the vesicular GABA uptake represented 33% of that in the 18-day embryo and 20% of adult levels. Thus the presynaptic GABAergic components developed according to similar schedules both in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Veratridine-induced release in vivo and in vitro of amino acids in the rabbit olfactory bulb

Free amino acids were studied in the olfactory bulb of the rabbit during basal conditions and veratridine-induced depolarization, in vitro with a tissue slice preparation and in vivo with a perfusion-dialysis technique. In vivo, basal extracellular concentrations of GABA, β-alanine and aspartate were low, while glutamine showed the highest level. The basal steady-state concentration ratio between the total tissue pool of free amino acids and the extracellular fluid was high for GABA, aspartate and glutamate, while low for glutamine and other ‘non-transmitter’ amino acids. Veratridine induced a marked TTX-sensitive release of GABA (40–50 times the control) both in vivo and in vitro. In vivo, the GABA release showed a peak during the first minutes of veratridine perfusion. The TTX-sensitive release of aspartate and glutamate, on the other hand, was approximately 5 times higher in vitro than in vivo. Furthermore, a prolonged response to veratridine was seen for glutamate and aspartate in vivo consisting of an early peak, followed by a sustained release. Taurine showed a time-delayed veratridine response, both in vivo and in vitro, whereas glutamine displayed a slow, TTX-sensitive decrease. No effect of veratridine was seen on β-alanine or carnosine-threonine levels.     

In vitro and in vivo studies of the anti-Xa activity of heparin

Several heparin preparations have been assayed using the Denson and Bonnar anti-Xa assay. The heat defibrination step involved in this assay has been shown to introduce discrepancies into the results, due mainly to co-precipitation of the heparin with fibrinogen. The amount of heparin lost is dependant on the molecular weight. The anti-Xa activity of ex vivo samples from subjects given heparin was much more resistant to loss during defibrination than in vitro samples, resulting in artificially high potency estimates. A modified assay has been proposed, omitting the defibrination step. The results provide further evidence that the anti-Xa activity observed after subcutaneous injection of heparin differs from that measured when the drug is added to plasma in vitro.