Cyclooxygenase inhibition limits blood-brain barrier disruption following intracerebral injection of tumor necrosis factor-alpha in the rat

Increased permeability of the blood-brain barrier (BBB) is important in neurological disorders. Neuroinflammation is associated with increased BBB breakdown and brain injury. Tumor necrosis factor (TNF)-alpha is involved in BBB injury and edema formation through a mechanism involving matrix metalloproteinase (MMP) up-regulation. There is emerging evidence indicating that cyclooxygenase (COX) inhibition limits BBB disruption following ischemic stroke and bacterial meningitis, but the mechanisms involved are not known. We used intracerebral injection of TNF-alpha to study the effect of COX inhibition on TNF-alpha-induced BBB breakdown, MMP expression/activity, and oxidative stress. BBB disruption was evaluated by the uptake of (14)C-sucrose into the brain and by magnetic resonance imaging utilizing gadolinium-diethylenetriaminepentaacetic acid as a paramagnetic contrast agent. Using selective inhibitors of each COX isoform, we found that COX-1 activity is more important than COX-2 in BBB opening. TNF-alpha induced a significant up-regulation of gelatinase B (MMP-9), stromelysin-1 (MMP-3), and COX-2. In addition, TNF-alpha significantly depleted glutathione as compared with saline. Indomethacin (10 mg/kg i.p.), an inhibitor of COX-1 and COX-2, reduced BBB damage at 24 h. Indomethacin significantly attenuated MMP-9 and MMP-3 expression and activation and prevented the loss of endogenous radical scavenging capacity following intracerebral injection of TNF-alpha. Our results show for the first time that BBB disruption during neuroinflammation can be significantly reduced by administration of COX inhibitors. Modulation of COX in brain injury by COX inhibitors or agents modulating prostaglandin E(2) formation/signaling may be useful in clinical settings associated with BBB disruption.     

Phosphodiesterase isozymes involved in regulation of formula secretion in isolated mouse stomach in vitro

(+/-)-(E)-4-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide] (NOR-3), a nitric-oxide (NO) donor, is known to increase HCO(3)(-) secretion in rat stomachs, intracellularly mediated by cGMP; yet, there is no information about the phosphodiesterase (PDE) isozyme involved in this process. We examined the effects of various isozyme-selective PDE inhibitors on the secretion of HCO(3)(-) in the mouse stomach in vitro and the type(s) of PDE isozymes involved in the response to NO. The gastric mucosa of DDY mice was stripped of the muscle layer and mounted on an Ussing chamber. HCO(3)(-) secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. NOR-3, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), and various PDE inhibitors were added to the serosal side. Vinpocetine (PDE1 inhibitor) or zaprinast (PDE5 inhibitor) was also added serosally 30 min before NOR-3 or 8-Br-cGMP. Both NOR-3 and 8-Br-cGMP stimulated HCO(3)(-) secretion in a dose-dependent manner, and the response to NOR-3 was significantly inhibited by methylene blue. Likewise, the secretion induced by NOR-3 or 8-Br-cGMP was significantly attenuated by 6-((2S,3S)-3-(4-chloro-2-methylphenylsulfonylaminomethyl)-bicyclo(2.2.2)octan-2-yl)-5Z-hexenoic acid (ONO-8711), the PGE receptor (EP)1 antagonist, as well as indomethacin and potentiated by both vinpocetine and zaprinast at doses that had no effect by themselves on the basal secretion, whereas other subtype-selective PDE inhibitors had no effect. NOR-3 increased the mucosal PGE(2) content in a methylene blue-inhibitable manner. These results suggest that NO stimulates gastric HCO(3)(-) secretion mediated intracellularly by cGMP and modified by both PDE1 and PDE5, and this response is finally mediated by endogenous PGE(2) via the activation of EP1 receptors.     

In vivo imaging of functional disruption, recovery and alteration in rat olfactory circuitry after lesion

Compensatory changes following disruption of neuronal circuitry have been indicated by previous imaging studies of stroke and other brain injury, but evidence of the pathways involved in such dynamic changes has not been shown in vivo. We imaged rats before and after lesion-induced disruption of the lateral olfactory tract to investigate the subsequent recovery and/or reorganization of functional neuronal circuitry. Serial magnetic resonance imaging was performed following intranasal administration of a paramagnetic track tracer Mn(2+). Images were analyzed using statistical mapping techniques in the stereotactic coordinate system. At 1 week post-lesion, Mn(2+) transport caudal to lesion was reduced as expected, and more importantly, increased transport through the anterior commissure was seen. At 4 weeks post-lesion, there was recovery of transport caudal to lesion, and increased transport through the anterior commissure extended to the contralateral olfactory cortex. Correlation analysis of regional Mn(2+) transport indicated that contralateral enhancement was not simply due to septal window spillover. This study demonstrates for the first time in vivo evidence of compensatory changes in functional neuronal activity to a contralateral pathway through the commissure following brain injury.     

Pepsin output after disruption of the human gastric mucosal barrier by taurocholic acid.

Test solutions containing 10 mM taurocholic acid in 160 mM HCl were instilled into the stomachs of healthy volunteers to disrupt the gastric mucosal barrier. Although significant back diffusion of hydrogen ions and exsorption of sodium ions occurred, there was no significant increase in pepsin output compared to control studies with 160 mm HCl alone. Our data suggest that 160 mM HCl produces a many-fold increase in basal pepsin output. In the innervated stomach of man disruption of the gastric mucosal barrier by taurocholic acid in acid solution is not accompanied by increased pepsin secretion compared to that produced by 160 mM HCl alone.     

脑缺血再灌注后基质金属蛋白酶-9的早期表达及对血脑屏障的破坏

目的研究大鼠短暂性脑缺血再灌注对脑组织基质金属蛋白酶-9(MMPs)表达的影响及对血脑屏障的破坏程度，探讨两者之间的关系以及在脑缺血损伤中所起的作用。方法通过制作大鼠脑缺血／再灌注模型，观察脑缺血后不同再灌注时间对脑含水量、血脑屏障通透性及MMPs表达的影响。结果脑缺血再灌注6h时，大鼠脑组织即有含水量及血脑屏障通透性增加，并随着时间推移而呈上升趋势。MMPs主要表达在大鼠缺血侧脑半球内的缺血神经元、血管内皮细胞及嗜中性粒细胞等细胞胞浆中，其表达含量于缺血再灌注6h后开始增加，并于再灌注1～2d时达到峰值，而该组大鼠缺血对侧脑半球组织内未见MMPs表达；假手术组大鼠亦未发现MMPs表达。结论MMPs的早期表达与血脑屏障通透性的改变密切相关．提示MMPs可能参与脑梗死后血管源性水肿的早期形成，从而加重脑缺血再灌注损伤。     

Astrocyte-derived VEGF-A drives blood-brain barrier disruption in CNS inflammatory disease

In inflammatory CNS conditions such as multiple sclerosis (MS), current options to treat clinical relapse are limited, and more selective agents are needed. Disruption of the blood-brain barrier (BBB) is an early feature of lesion formation that correlates with clinical exacerbation, leading to edema, excitotoxicity, and entry of serum proteins and inflammatory cells. Here, we identify astrocytic expression of VEGF-A as a key driver of BBB permeability in mice. Inactivation of astrocytic Vegfa expression reduced BBB breakdown, decreased lymphocyte infiltration and neuropathology in inflammatory and demyelinating lesions, and reduced paralysis in a mouse model of MS. Knockdown studies in CNS endothelium indicated activation of the downstream effector eNOS as the principal mechanism underlying the effects of VEGF-A on the BBB. Systemic administration of the selective eNOS inhibitor cavtratin in mice abrogated VEGF-A-induced BBB disruption and pathology and protected against neurologic deficit in the MS model system. Collectively, these data identify blockade of VEGF-A signaling as a protective strategy to treat inflammatory CNS disease.     

Signal transduction pathways involved in the mechanical responses to protease-activated receptors in rat colon

Recording simultaneously in vitro the changes of endoluminal pressure (index of circular muscle activity) and isometric tension (index of longitudinal muscle activity), we examined the mechanisms responsible for the apamin-sensitive relaxant and contractile responses induced by protease-activated receptor (PAR)-1 and PAR-2 activating peptides, SFLLRN-NH2 and SLIGRL-NH2, respectively, in rat colon. In the circular muscle, the inhibitory effects of SFLLRN-NH2 and SLIGRL-NH2 were significantly reduced by ryanodine, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, but unaffected by 1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF109203X), a protein kinase C (PKC) inhibitor, or genistein, a tyrosine kinase inhibitor. In the longitudinal muscle, the contractile responses to SFLLRN-NH2 and SLIGRL-NH2 were significantly reduced by nifedipine, an L-type calcium channel blocker, ryanodine, GF109203X, genistein, and abolished by U73122. The effects of genistein were additive with GF109203X but not with nifedipine. In the longitudinal muscle, the relaxant responses to the highest concentrations of SFLLRN-NH2 and SLIGRL-NH2 were abolished by nifedipine, reduced by genistein, and unaffected by ryanodine or GF109203X. In conclusion, influx of extracellular Ca2+ through L-type voltage-dependent channels or release of Ca2+ from intracellular stores are determining for the opening of the apamin-sensitive K+ channels responsible for longitudinal muscle relaxation or circular muscle inhibitory response, respectively, in rat colon. The longitudinal muscle contraction is mediated by activation of PLC; PKC and tyrosine kinase are involved in the cascade process, playing a parallel role. Indeed, tyrosine kinase and L-type Ca2+ channels would act sequentially. The influx of Ca2+ in turn would cause release of Ca2+ from sarcoplasmic reticulum.     

Selective inhibition by chloramphenicol of cytochrome P-450 isozymes in rat lung and liver involved in the hydroxylation of n-hexane

Treatment of rats with chloramphenicol causes a dose-dependent and regioselective inhibition of the metabolism of the organic solvent n-hexane in both liver and lung microsomes. A dose of chloramphenicol of 100 mg kg-1 administered i.v. or i.p. results in more than 50% inhibition of 2-hexanol formation catalyzed by microsomes from both organs, but causes no inhibition of 1-hexanol formation. The effects of chloramphenicol on 3-hexanol formation are somewhat organ-specific. In the liver 3-hexanol formation is inhibited to almost the same extent as 2-hexanol formation, whereas in the lung the inhibition of the formation of 3-hexanol is markedly less. Phenobarbital induces n-hexane metabolism in the liver but not the lung, but decreases the inhibitory potency of chloramphenicol toward both organs. In vitro chloramphenicol causes both reversible and irreversible inhibition of 2-hexanol formation in control lung microsomes. The irreversible inhibition is accompanied by the covalent binding of metabolites of chloramphenicol to the lung microsomes. The covalent binding is completely inhibited by antibodies to the major phenobarbital-induced isozyme of rat liver cytochrome P-450. These antibodies also cause more than 90% inhibition of 2-hexanol formation by lung microsomes. The results suggest that chloramphenicol acts as a selective suicide substrate of a constitutive isozyme of rat lung cytochrome P-450 involved in the 2-hydroxylation of n-hexane.     

Pharmacokinetics of methotrexate in cerebrospinal fluid and serum after osmotic blood-brain barrier disruption in patients with brain lymphoma

OBJECTIVE: To evaluate the pharmacokinetics of methotrexate in ventricular cerebrospinal fluid and serum after osmotic blood-brain barrier disruption and intra-arterial administration compared with intravenous or simple intra-arterial infusion in patients with primary central nervous system lymphoma. METHODS: Serum and ventricular cerebrospinal fluid were sampled after methotrexate administration in 12 patients. Blood-brain barrier disruption was induced on 2 sequential days by mannitol (25%) infusion delivered to the vertebral or internal carotid artery territories followed by intra-arterial methotrexate (dose, 1.4 g/m2; 47 treatments). Sixteen treatments were given without barrier disruption by intravenous (3.5 g/m2; nine treatments) or intra-arterial (2.8 g/m2; seven treatments) infusion. RESULTS: Ventricular cerebrospinal fluid-methotrexate peak levels after blood-brain barrier disruption of the vertebral and the internal carotid arteries territories were 19.3 +/- 2.9 and 8.5 +/- 0.7 micromol/L (P < .001), and the area under the curve from time 0 to infinity was 178.0 +/- 21.3 and 110.0 +/- 12.4 [micromol/L x h, respectively (P < .01). No significant differences were observed in serum levels. After intra-arterial infusion was performed without disruption, the serum peak level was higher than that achieved by intravenous treatment (518.2 +/- 67.7 versus 180.6 +/- 31.8 micromol/L; P < .001). No differences were observed in cerebrospinal fluid concentrations, which dropped below 1 micromol/L at 6 hours. The cerebrospinal fluid/serum ratio [AUC(%)] of methotrexate after blood-brain barrier disruption was three to four times greater than that by systemic administration. CONCLUSION: Enhanced methotrexate delivery to the central nervous system can be attained by intra-arterial administration combined with osmotic disruption of the blood-brain barrier compared with simple intra-arterial or intravenous administration.     

Somatostatin-gastrin interactions in the rat stomach

Low concentrations of somatostatin and gastrin within or slightly above the range of physiologically circulating levels were perfused in the isolated, vascularly perfused rat stomach preparation. Somatostatin at 10 and 50 pg/ml significantly inhibited acetylcholine-stimulated gastrin secretion by 26% and 45%, respectively, whereas perfusion of 50 and 500 pg/ml exogenous gastrin did not modify gastric somatostatin secretion. Perfusion of somatostatin-antiserum significantly increased gastrin release by 235%. It is concluded that (1) somatostatin is a powerful inhibitor of the gastrin cell under in vitro conditions; the data are in accordance with a concept that endogenous somatostatin could act as a true hormone; (2) the secretory activity of the somatostatin cell is not significantly affected by circulating gastrin.     

Lipid solubilization during bile salt-induced esophageal mucosal barrier disruption in the rabbit

Bile salts in the gastric contents are one of the harmful agents that may contribute to the mucosal injury found in clinical reflux esophagitis. To clarify the mechanism by which bile salts can injure the esophageal mucosa, we studied the effects of exposure to the bile salts taurocholate and deoxycholate in an in vivo perfused rabbit model of esophagitis. Specifically we examined the roles of accumulation of bile salt by the mucosa and solubilization of mucosal lipid into the lumen during bile salt-induced mucosal injury. Results showed a consistent association between bile salt accumulation by the esophageal mucosa and bile salt-induced disruption of the physiologic barrier to diffusion. Under certain conditions, bile salts caused barrier disruption with no release of lipid from the mucosa. Whenever exposure to bile salt did cause release of mucosal lipid into the lumen, a significant amount of morphologic injury was also observed by light microscopy. The findings suggest that the presence of bile salt in the mucosa is an important aspect of the mechanism of bile salt-induced barrier disruption. Solubilization of mucosal lipids into the lumen occurs when bile salts cause an injury that is severe enough to result in light microscopic evidence of morphologic damage.     

MRI-guided targeted blood-brain barrier disruption with focused ultrasound: histological findings in rabbits

Focused ultrasound offers a method to disrupt the blood-brain barrier (BBB) noninvasively and reversibly at targeted locations. The purpose of this study was to test the safety of this method by searching for ischemia and apoptosis in areas with BBB disruption induced by pulsed ultrasound in the presence of preformed gas bubbles and by looking for delayed effects up to one month after sonication. Pulsed ultrasound exposures (sonications) were performed in the brains of 24 rabbits under monitoring by magnetic resonance imaging (MRI) (ultrasound: frequency = 1.63 MHz, burst length = 100 ms, PRF = 1 Hz, duration = 20 s, pressure amplitude 0.7 to 1.0 MPa). Before sonication, an ultrasound contrast agent (Optison, GE Healthcare, Milwaukee, WI, USA) was injected IV. BBB disruption was confirmed with contrast-enhanced MR images. Whole brain histologic examination was performed using haematoxylin and eosin staining for general histology, vanadium acid fuchsin-toluidine blue staining for ischemic neurons and TUNEL staining for apoptosis. The main effects observed were tiny regions of extravasated red blood cells scattered around the sonicated locations, indicating affected capillaries. Despite these vasculature effects, only a few cells in some of the sonicated areas showed evidence for apoptosis or ischemia. No ischemic or apoptotic regions were detected that would indicate a compromised blood supply was induced by the sonications. No delayed effects were observed either by MRI or histology up to 4 wk after sonication. Ultrasound-induced BBB disruption is possible without inducing substantial vascular damage that would result in ischemic or apoptotic death to neurons. These findings indicate that this method is safe for targeted drug delivery, at least when compared with the currently available invasive methods.     

Systemic delivery of antagomirs during blood-brain barrier disruption is disease-modifying in experimental epilepsy

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Contractile serotonergic receptor in rat stomach fundus

Serotonin (5HT) is a potent agonist in contracting the rat stomach fundus although the nature of the receptor mediating the response has not been established. The present study was designed to explore the possibility that 5HT-induced contractions in the rat stomach fundus were mediated by interaction with receptors identical with either 5HT1A, 5HT1B or 5HT1C binding sites or 5HT3 receptors. Contractile concentration-response curves for several 5HT agonists [5-carboxamidotryptamine, TR3369, MK212, quipazine, RU 24969, 8-hydroxy-2-(di-n-propylamino)tetralin, TVXQ7821 and BEA 1654CL] were obtained in the rat stomach fundus. However, neither the potency nor maximum response of these agonists in contracting the rat stomach fundus correlated with the affinity of agonists at 5HT1A, 5Ht1B or 5HT1C binding sites. These agonists were interacting with 5HT receptors in the fundus based on the ability of 1-(1-naphthyl) piperazine (10(-7) M) to antagonize the contractile response of the relatively potent agonists. TVXQ7821 and BEA 1654Cl did not produce a marked contractile response in the fundus and also did not antagonize the contractile response to 5HT, suggesting that these agents have little, if any, affinity for the serotonergic receptor-mediating contraction in the fundus. The putative 5HT1A-selective receptor antagonists, WB4101 and spiroxatrine, did not block 5HT-induced contractions in the rat stomach fundus in concentrations consistent with their affinity at 5HT1A binding sites. The nonselective 5HT1A and 5HT1B receptor antagonist, cyanopindolol, also did not block 5HT-induced contractions in the rat stomach fundus.(ABSTRACT TRUNCATED AT 250 WORDS)