Involvement of tumor necrosis factor alpha and interleukin-1beta in enhancement of pentylenetetrazole-induced seizures caused by Shigella dysenteriae.
Neurologic manifestations, mainly convulsions, are the most frequent extraintestinal complications of shigellosis. We used an animal model to study the roles of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in Shigella-related seizures. Administration of Shigella dysenteriae 60R sonicate enhanced the sensitivity of mice to the proconvulsant pentylenetetrazole (PTZ) within 7 h. This was indicated by a significantly higher mean convulsion score and an increased number of mice responding with clonic-tonic seizures in the Shigella-pretreated group. Preinjection of mice with anti-murine TNF-alpha (anti-mTNF-alpha) or anti-murine IL-1beta (anti-mIL-1beta) 30 min prior to administration of Shigella sonicate abolished their enhanced response to PTZ at 7 h. Mean convulsion scores were reduced by anti-mTNF-alpha from 1.2 to 0.8 (P = 0.017) and by anti-mIL-1beta from 1.3 to 0.7 (P = 0.008). Preinjection of anti-mTNF-alpha also reduced the percentage of mice responding with clonic-tonic seizures, from 48 to 29% (P = 0.002), and preinjection of anti-mIL-1beta reduced it from 53 to 21% (P = 0. 012). Neutralization of TNF-alpha or IL-1beta did not protect the mice from death due to S. dysenteriae 60R. These findings indicate that TNF-alpha and IL-1beta play a role in the very early sensitization of the central nervous system to convulsive activity after S. dysenteriae administration. Similar mechanisms may trigger neurologic disturbances in other infectious diseases. (+info)
Drug-protein binding and blood-brain barrier permeability.
The permeability surface area (PS) product, an index of permeability of the blood-brain barrier (BBB), was measured by using the in situ perfusion method. In the cerebral circulation, the fraction of drug that permeates into the brain through the BBB is not only the unbound fraction but also the fraction dissociated from the protein in the perfusate. The sum of these two fractions, the apparent exchangeable fraction, was estimated by fitting the parameters of the BBB permeability under the condition of varying BSA concentrations in the perfusate. The unbound fraction of drugs in a buffer containing 0.5 mM BSA was measured by using the ultrafiltration method in vitro, and the apparent exchangeable fraction was measured in vivo by using the intracarotid artery injection method. The apparent exchange fraction was 100% for S-8510, 96.5% for diazepam, 90.9% for caffeine, 38.3% for S-312-d, 33.1% for propranolol, and 6.68% for (+)-S-145 Na, and each of these was higher than the corresponding unbound fraction in vitro in all drugs. The apparent exchangeable fractions, for example, were 8 times higher for diazepam and 38 times for S-312-d than the unbound fractions in vitro. The apparent exchangeable fraction of drugs was also estimated from the parameters obtained with the perfusion method. Because drugs can be infused for an arbitrary length of time in the perfusion method, substances with low permeability can be measured. The apparent exchangeable fractions obtained with this method were almost the same as those obtained with the intracarotid artery injection method. (+info)
Novel, highly lipophilic antioxidants readily diffuse across the blood-brain barrier and access intracellular sites.
In an accompanying article, an in vitro assay for permeability predicts that membrane-protective, antioxidant 2,4-diamino-pyrrolo[2, 3-d]pyrimidines should have improved blood-brain barrier (BBB) permeation over previously described lipophilic antioxidants. Using a first-pass extraction method and brain/plasma quantification, we show here that two of the pyrrolopyrimidines, one of which is markedly less permeable, readily partition into rat brain. The efficiency of extraction was dependent on serum protein binding, and in situ efflux confirms the in vitro data showing that PNU-87663 is retained in brain longer than PNU-89843. By exploiting inherent fluorescence properties of PNU-87663, its distribution within brain and within cells in culture was demonstrated using confocal scanning laser microscopy. PNU-87663 rapidly partitioned into the cell membrane and equilibrates with cytoplasmic compartments via passive diffusion. Although partitioning of PNU-87663 favors intracytoplasmic lipid storage droplets, the compound was readily exchangeable as shown by efflux of compound from cells to buffer when protein was present. The results demonstrated that pyrrolopyrimidines were well suited for quickly accessing target cells within the central nervous system as well as in other target tissues. (+info)
Inhibition by lead of production and secretion of transthyretin in the choroid plexus: its relation to thyroxine transport at blood-CSF barrier.
Long-term, low-dose Pb exposure in rats is associated with a significant decrease in transthyretin (TTR) concentrations in the CSF. Since CSF TTR, a primary carrier of thyroxine in brain, is produced and secreted by the choroid plexus, in vitro studies were conducted to test whether Pb exposure interferes with TTR production and/or secretion by the choroid plexus, leading to an impaired thyroxine transport at the blood-CSF barrier. Newly synthesized TTR molecules in the cultured choroidal epithelial cells were pulse-labeled with [35S]methionine. [35S]TTR in the cell lysates and culture media was immunoprecipitated and separated by SDS-PAGE, and quantitated by autoradiography and liquid scintillation counting. Pb treatment did not significantly alter the protein concentrations in the culture, but inhibited the synthesis of total [35S]TTR (cells + media), particularly during the later chase phase. Two-way ANOVA of the chase phase revealed that Pb exposure (30 microM) significantly suppressed the rate of secretion of [35S]TTR compared to the controls (p < 0.05). Accordingly, Pb treatment caused a retention of [35S]TTR by the cells. In a two-chamber transport system with a monolayer of epithelial barrier, Pb exposure (30 microM) reduced the initial release rate constant (kr) of [125I]T4 from the cell monolayer to the culture media and impeded the transepithelial transport of [125I]T4 from the basal to apical side of epithelial cells by 27%. Taken together, these in vitro data suggest that sequestration of Pb in the choroid plexus hinders the production and secretion of TTR by this tissue. Consequently, this may alter the transport of thyroxine across this blood-CSF barrier. (+info)
Receptor-mediated transcytosis of lactoferrin through the blood-brain barrier.
Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation; it accumulates in the brain during neurodegenerative disorders. Before determining Lf function in brain tissue, we investigated its origin and demonstrate here that it crosses the blood-brain barrier. An in vitro model of the blood-brain barrier was used to examine the mechanism of Lf transport to the brain. We report that differentiated bovine brain capillary endothelial cells exhibited specific high (Kd = 37.5 nM; n = 90,000/cell) and low (Kd = 2 microM; n = 900,000 sites/cell) affinity binding sites. Only the latter were present on nondifferentiated cells. The surface-bound Lf was internalized only by the differentiated cell population leading to the conclusion that Lf receptors were acquired during cell differentiation. A specific unidirectional transport then occurred via a receptor-mediated process with no apparent intraendothelial degradation. We further report that iron may cross the bovine brain capillary endothelial cells as a complex with Lf. Finally, we show that the low density lipoprotein receptor-related protein might be involved in this process because its specific antagonist, the receptor-associated protein, inhibits 70% of Lf transport. (+info)
Nonsaturable entry of neuropeptide Y into brain.
Neuropeptide Y (NPY) is found and is active both in the periphery and brain, but its crossing of the blood-brain barrier (BBB) in either direction has not been measured. We used multiple time-regression analysis to determine that radioactively labeled NPY injected intravenously entered the brain much faster than albumin, with an influx constant of 2.0 x 10(-4) ml. g. -1. min-1. However, this rate of entry was not significantly changed by injection of 10 microgram/mouse of excess NPY, by leptin, or by food deprivation. HPLC showed that most of the NPY entering the brain was intact, and capillary depletion with and without washout showed that the NPY did not remain bound to endothelial cells or associated with vascular elements. Perfusion in a blood-free solution eliminated binding to serum proteins as an explanation for the lack of saturation. Efflux of labeled NPY from the brain occurred at the same rate as albumin, reflecting the normal rate of reabsorption of cerebrospinal fluid. Thus NPY can readily enter the brain from blood by diffusion across the BBB. (+info)
Selective delivery of herpes virus vectors to experimental brain tumors using RMP-7.
RMP-7, a bradykinin analog, has been shown to selectively open the blood-tumor barrier for the delivery of chemotherapeutic drugs to brain tumors. In contrast to bradykinin, RMP-7 has no hypotensive effects and has been approved for human use. This study was initiated to determine whether RMP-7 would open the blood-tumor barrier to virus vectors encoding tumor-killing genes in an experimental model. The herpes virus vector used, hrR3, which encodes virus thymidine kinase gene and the lacZ reporter gene, is defective in a gene encoding ribonucleotide reductase, replicates selectively in dividing tumor cells and not in postmitotic neural cells. It was determined that an optimum dose of RMP-7 (1.5-3.0 microg/kg over 10-15 minutes) enhanced viral delivery to brain tumors in rats bearing intracranial 9 L gliosarcomas when infused through the carotid artery immediately prior to virus vector application. Maximum expression of the lacZ reporter gene occurred at 3 days after intracarotid infusion. By 8 days, transgene expression was largely confined to tumor foci away from the main tumor mass. Viral delivery was essentially specific to tumor cells, with little transgene expression elsewhere in the brain. Minimal uptake and pathology was noted in the kidney, spleen, and liver. These findings indicate that intracarotid delivery of RMP-7 can augment the selective delivery of virus vectors to brain tumors in an experimental rat model, with the potential for application to human brain tumors. (+info)
Orexin A but not orexin B rapidly enters brain from blood by simple diffusion.
We determined the ability of orexin A and orexin B, recently discovered endogenous appetite enhancers, to cross the blood-brain barrier (BBB) of mice. Multiple time-regression analysis showed that an i.v. bolus of 125I-orexin A rapidly entered the brain from the blood, with an influx rate (Ki = 2.5 +/- 0.3 x 10(-4) ml/g.min) many times faster than that of the 99mTc-albumin control. This relatively rapid rate of entry was not reduced by administration of excess orexin A (or leptin) or by fasting for 22 h, even when penetration into only the hypothalamus was measured. Lack of saturability also was shown by perfusion in blood-free buffer. HPLC revealed that most of the injected 125I-orexin A reached the brain as intact peptide. Capillary depletion studies showed that the administered peptide did not remain bound to the endothelial cells comprising the BBB but reached the brain parenchyma. Efflux of 125I-orexin A from the brain occurred at the same rate as 99mTc-albumin. The octanol/buffer partition coefficient of 0.232 showed that orexin A was highly lipophilic, whereas the value for orexin B was only 0.030. Orexin B, moreover, was rapidly degraded in blood, so no 125I-orexin B could be detected in intact form in brain when injected peripherally. Thus, although orexin B is rapidly metabolized in blood and has low lipophilicity, orexin A rapidly crosses the BBB from blood to reach brain tissue by the process of simple diffusion. (+info)