Vascular changes play a role in the pathogenesis of necrotizing enterocolitis in asphyxiated newborn pigs.

Necrotizing enterocolitis (NEC) is the most common acquired gastrointestinal emergency in neonates. We have developed an animal model of NEC in asphyxiated newborn pigs and investigated the effects of asphyxia on blood flow in superior mesenteric artery and abdominal aorta, cardiovascular data, arterial acid-base and blood gas parameters, and endothelial cytoskeletal structure in mesenteric microvasculature. Anesthetized, mechanically ventilated newborn pigs were included in two groups: piglets underwent severe asphyxia, and sham-operated control animals. A cardiovascular and metabolic failure developed in asphyxiated piglets approximately 1 h after the induction: severe hypotension and bradyarrhythmia were seen and significant reductions of the blood flow were measured in the superior mesenteric artery and abdominal aorta during the critical phase. Rearrangement of cytoskeletal actin structure corresponding to enhanced vascular permeability was seen with bodipy phallacidin in mesenterial endothelium of asphyxiated piglets after a 24-h recovery period. In conclusion, severe vasomotor changes during asphyxia may result in mesenteric endothelial dysfunction implicated in increased vascular permeability, edema formation, and development of NEC in asphyxiated piglets.

Chronic adrenomedullin treatment improves blood-brain barrier function but has no effects on expression of tight junction proteins.

We previously found that the production of adrenomedullin (AM) is one magnitude higher in cerebral endothelial cells (CECs) than in the peripheral endothelium and the AM concentration in the cerebral circulation is significantly higher than in other tested parts of the circulation. We also showed that CECs express AM receptors, and AM as an autocrine hormone is important to regulate the intracellular cAMP level in CECs. Further we reported that acute AM treatment has cAMP-like effects on specific BBB functions: AM decreased endothelial fluid phase endocytosis, activated the P-glycoprotein, increased transendothelial electrical resistance (TEER) and reduced endothelial permeability for sodium fluorescein, which suggests a tightening of intercellular junctions. In the present study, we found chronic AM exposure also increased TEER. In contrast, we could not detect significant effect of AM on the expression of tight junction proteins (claudin-1, occludin and zonula occludens-1). While not affecting expression of tight junction proteins, chronic AM treatment may influence the localization of these proteins which has been reported to correlate with functional changes of the BBB without a change in protein expression.

Cerebral endothelial cells are a major source of adrenomedullin.

Adrenomedullin is a peptide hormone with multifunctional biological properties. Its most characteristic effects are the regulation of circulation and the control of fluid and electrolyte homeostasis through peripheral and central nervous system actions. Although adrenomedullin is a vasodilator of cerebral vasculature, and it may be implicated in the pathomechanism of cerebrovascular diseases, the source of adrenomedullin in the cerebral circulation has not been investigated thus far. We measured the secretion of adrenomedullin by radioimmunoassay and detected adrenomedullin mRNA expression by Northern blot analysis in primary cultures of rat cerebral endothelial cells (RCECs), pericytes and astrocytes. We also investigated the expression of specific adrenomedullin receptor components by reverse transcriptase-polymerase chain reaction and intracellular cAMP concentrations in RCECs and pericytes. RCECs had approximately one magnitude higher adrenomedullin production (135 +/- 13 fmol/10(5) cells per 12 h; mean +/- SD, n = 10) compared to that previously reported for other cell types. RCECs secreted adrenomedullin mostly at their luminal cell membrane. Adrenomedullin production was not increased by thrombin, lipopolysaccharide or cytokines, which are known inducers of adrenomedullin release in peripheral endothelial cells, although it was stimulated by astrocyte-derived factors. Pericytes had moderate, while astrocytes had very low basal adrenomedullin secretion. In vivo experiments showed that adrenomedullin plasma concentration in the jugular vein of rats was approximately 50% higher than that in the carotid artery or in the vena cava. Both RCECs and pericytes, which are potential targets of adrenomedullin in cerebral microcirculation, expressed adrenomedullin receptor components, and exhibited a dose-dependent increase in intracellular cAMP concentrations after exogenous adrenomedullin administration. Antisense oligonucleotide treatment significantly reduced adrenomedullin production by RCECs and tended to decrease intraendothelial cAMP concentrations. These findings may suggest an important autocrine and paracrine role for adrenomedullin in the regulation of cerebral circulation and blood-brain barrier functions. Cerebral endothelial cells are a potential source of adrenomedullin in the central nervous system, where adrenomedullin can also be involved in the regulation of neuroendocrine functions.

Adrenomedullin regulates blood-brain barrier functions in vitro.

Adrenomedullin (AM) is an important vasodilator in cerebral circulation, and cerebral endothelial cells are a major source of AM. This in vitro study aimed to determine the AM-induced changes in blood-brain barrier (BBB) functions. AM administration increased, whereas AM antisense oligonucleotide treatment decreased transendothelial electrical resistance. AM incubation decreased BBB permeability for sodium fluorescein (mol. wt 376 Da) but not for Evan's blue albumin (mol. wt 67 kDa), and it also attenuated fluid-phase endocytosis. AM treatment resulted in functional activation of P-glycoprotein efflux pump in vitro. Our results indicate that AM as an autocrine mediator plays an important role in the regulation of BBB properties of the cerebral endothelial cells.

Asphyxia-induced release of alpha-melanocyte-stimulating hormone in newborn pigs.

Asphyxia and reperfusion induced changes in the plasma and cerebrospinal fluid (CSF) concentrations of alpha-melanocyte-stimulating hormone (alpha-MSH) were studied in newborn pigs using a specific radioimmunoassay technique. Cardiovascular and metabolic failure induced by neonatal asphyxia resulted in a 3-fold, significant (P < 0.05) increase in plasma alpha-MSH levels, whereas the hormone concentration in CSF was significantly (P < 0.05) reduced by 50% during postasphyxial reperfusion. Our data indicate an asphyxia-induced release of alpha-MSH, and suggest a discordant regulation of plasma and CSF concentrations in newborn pigs.

Tissue plasminogen activator inhibits P-glycoprotein activity in brain endothelial cells.

Tissue plasminogen activator (0.01-30 microgram/ml) dose-dependently inhibited the functional activity of P-glycoprotein, assessed by rhodamine 123 accumulation in GP8 immortalized rat brain endothelial cells, but this effect was unrelated to its proteolytic activity. Elevation of intra-endothelial cyclic AMP concentration and stimulation of protein kinase C increased P-glycoprotein activity in GP8 cells and also attenuated the tissue plasminogen activator-induced inhibition.

Adrenomedullin in the cerebral circulation.

The central nervous system requires an effective autoregulation of cerebral circulation in order to meet the critical and unusual demands of the brain. In addition, cerebral microvessels has a unique feature, the formation of the blood-brain barrier, which contributes to the stability of the brain parenchymal microenvironment. Many factors are known to be involved in the regulation of cerebral circulation and blood-brain barrier functions. In the last few years a new potential candidate, adrenomedullin, a hypotensive peptide was added to this list. Adrenomedullin has a potent vasodilator effect on the cerebral vasculature, and it may be implicated in the pathologic mechanism of cerebrovascular diseases. In this review, we describe current knowledge about the origin and possible role of adrenomedullin in the regulation of cerebral circulation and blood-brain barrier functions.

PrP fragment 106-126 is toxic to cerebral endothelial cells expressing PrP(C).

A hydrophobic, fibrillogenic peptide fragment of human prion protein (PrP106-126) had in vitro toxicity to neurons expressing cellular prion protein (PrP(C)). In this study, we proved that primary cultures of mouse cerebral endothelial cells (MCEC) express PrP(C). Incubation of MCEC with PrP106-126 (25-200 microM) caused a dose-dependent toxicity assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase release, bis-benzimide staining for nuclear morphology, and trypan blue exclusion test. Pentosan polysulphate (50-100 microg/ml), a drug effective in scrapie prophylaxis, dose-dependently attenuated the injury. MCEC cultures from mice homogenous for the disrupted PrP gene were resistant to the toxicity of PrP106-126. In conclusion, cerebral endothelium expressing PrP(C) may be directly damaged during spongiform encephalopathies.

Pentosan polysulfate regulates scavenger receptor-mediated, but not fluid-phase, endocytosis in immortalized cerebral endothelial cells.

1. Effects of pentosan polysulfate (PPS) and the structurally related sulfated polyanions dextran sulfate, fucoidan, and heparin on the scavenger receptor-mediated and fluidphase endocytosis in GP8 immortalized rat brain endothelial cells were investigated. 2. Using 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarboxyamine perchlorate-labeled acetylated low-density lipoprotein (DiI-AcLDL), we found a binding site with high affinity and low binding capacity, and another one with low affinity and high binding capacity. Increasing ligand concentrations could not saturate DiI-AcLDL uptake. DiI-AcLDL uptake, but not binding, was sensitive to pretreatment with filipin, an inhibitor of caveola formation. 3. PPS (20-200 microg/ml) significantly reduced the binding of DiI-AcLDL after coincubation for 3 hr, though this effect was less expressed after 18 hr. Among other polyanions, only fucoidan decreased the DiI-AcLDL binding after 3 hr, whereas dextran sulfate significantly increased it after 18 hr. PPS treatment induced an increase in DiI-AcLDL uptake, whereas other polysulfated compounds caused a significant reduction. 4. Fluid-phase endocytosis determined by the accumulation of Lucifer yellow was concentration and time dependent in GP8 cells. Coincubation with PPS or other sulfated polyanions could not significantly alter the rate of Lucifer yellow uptake. 5. In conclusion. PPS decreased the binding and increased the uptake of DiI-AcLDL in cerebral endothelial cells, an effect not mimicked by the other polyanions investigated.

Effects of N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine on the blood-brain barrier permeability in the rat.

Histamine plays a role in the regulation of the blood-brain barrier function. In this study, effects of N, N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine (DPPE), an intracellular histamine binding site antagonist on the cerebrovascular permeability were investigated in control and post-ischemic male Wistar rats. Intravenous administration of DPPE, in a dose of 1 and 5 mg/kg, was not followed by any major clinical change, but 20 mg/kg proved to be toxic. A significantly (P<0.05) increased permeability for sodium fluorescein (MW=376) was seen in hippocampus, striatum, and cerebellum, but not in parietal cortex, of rats 2 h after the injection of 5 mg/kg DPPE, whereas no increase was measured later. There was a more intense (5- to 12-fold) and prolonged elevation in Evan's blue-labeled albumin (MW=67,000) extravasation 2, 4, and 8 h after 5 mg/kg DPPE administration in each brain region. In parietal cortex, a dose-dependent increase in albumin extravasation developed 4 h after intravenous injection of 1, 5, and 20 mg/kg DPPE, but doses applied resulted in no significant change in sodium fluorescein permeability. Cerebral ischemia-reperfusion evoked by four-vessel occlusion caused a significant (P<0.05) increase in the permeability for albumin in each region, but few changes in that of sodium fluorescein. DPPE treatment failed to prevent the ischemia-reperfusion-induced changes in the blood-brain barrier permeability. In conclusion, DPPE induced an increased permeability in the rat, which supports a role for histamine, as an intracellular messenger, in the regulation of the blood-brain barrier characteristics.

4-(2-Aminoethyl)benzenesulfonyl fluoride attenuates tumor-necrosis-factor-alpha-induced blood-brain barrier opening.

The effect of serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) was investigated on the prevention of tumor-necrosis-factor-alpha (TNF-alpha)-induced blood-brain barrier opening. TNF-alpha (10,000 IU) was injected intracarotidly to newborn pigs pretreated with 0, 2.4, 4.8, 9.6 and 19.2 mg/kg AEBSF (n = 6 in each group). AEBSF dose-dependently inhibited the TNF-alpha-induced increase in the blood-brain barrier permeability for sodium fluorescein (MW = 376) in all of the five brain regions examined, while only 19.2 mg/kg AEBSF could significantly (P < 0.05) decrease the change in Evan's blue-albumin (MW = 67,000) transport in two regions. In conclusion, AEBSF attenuates vasogenic brain edema formation.

Differential regulation of vasopressin gene expression in the hypothalamus of endotoxin-treated 14-day-old rat.

The E. coli endotoxin 0111 B4, a lipopolysaccharide (LPS), in a dose of 200 ng/kg body weight/50 microl artificial cerebrospinal fluid (CSF) was given intracisternally to 14-day-old rats. Four hours later CSF, blood and urine were sampled, and consecutive brain sections from the hypothalamic area of the brain were prepared for in situ hybridization. The LPS treatment resulted in a significant (p<0.001) pleocytosis and an elevation of the protein content of the CSF. There were no changes observed in the chemical parameters of the CSF, plasma, blood or urine, i.e. vasopressin (VP) levels, osmolality, Na+ and K+ concentrations, glucose level, pH, bicarbonate or PaCO2, PaO2 values. LPS injection, however, resulted in a significantly (p<0.01) increased VP mRNA level (121% of the control value) in the supraoptic nuclei (SON), but not in the paraventricular nuclei (PVN), as compared to controls. Our findings suggest an early effect of LPS on VP gene expression selectively in the SON of 14-days-old rats. This animal model might be suitable for studying the regulation of VP gene expression and the role of this peptide in the pathogenesis of bacterial meningitis in pediatric patients.

Cerebral ischemia reperfusion-induced vasogenic brain edema formation in rats: effect of an intracellular histamine receptor antagonist.

Resuscitation in pediatric emergency and some neurological interventions may result in ischemia reperfusion-induced cerebral injuries. Histamine is one of the well established mediators of cerebral swelling and H1- and H2-receptor antagonists could prevent the development of ischemic brain edema. In the present study, time-dependent changes in the blood-brain barrier (BBB) permeability were investigated in the cerebral cortex of male Wistar rats 1, 2, 4, 8, and 16 h after the beginning of post-ischemic reperfusion. Cerebral ischemia-reperfusion evoked by the 4-vessel occlusion model resulted in significant (p < 0.05) elevations in BBB permeability for albumin, but not for sodium fluorescein. Pre-treatment with a new intracellular histamine receptor antagonist could not prevent ischemic brain edema formation in that model. We conclude that experimental studies could help us to reveal the therapeutic role of histamine receptor antagonists during ischemic brain edema.

Experimental acute pancreatitis results in increased blood-brain barrier permeability in the rat: a potential role for tumor necrosis factor and interleukin 6.

Pancreatic encephalopathy is a severe complication of acute pancreatitis. Proinflammatory cytokines may play a role in the development of multi-organ failure during pancreatitis. In the present study, we measured the changes in the blood-brain barrier (BBB) permeability concomitantly with the determination of serum tumor necrosis factor (TNF) and interleukin-6 (IL-6) levels in rats before, as well as 6, 24 and 48 h after the beginning of intraductal taurocholic acid-induced acute pancreatitis. Cytokine concentrations were measured in bioassays with specific cell lines (WEHI-164 for TNF and B-9 for IL-6), while the BBB permeability was determined for a small (sodium fluorescein, molecular weight (MW) 376 Da), and a large (Evans' blue-albumin, MW 67000 Da) tracer by spectrophotometry in the parietal cortex, hippocampus, striatum, cerebellum and medulla of rats. The serum TNF level was significantly (P < 0.05) increased 6 and 24 h after the induction of pancreatitis, while the IL-6 level increased after 24 and 48 h. A significant (P < 0.05) increase in BBB permeability for both tracers developed at 6 and 24 h in different brain regions of animals with acute pancreatitis. We conclude that cytokines, such as TNF and IL-6, may contribute to the vasogenic brain edema formation during acute pancreatitis.