Penetration of verapamil across blood brain barrier following cerebral ischemia depending on both paracellular pathway and P-glycoprotein transportation

Background

Blood brain barrier (BBB) dysfunction is a common facet of cerebral ischemia, and the alteration of drug transporter, P-glycoprotein (P-gp), has been documented.

Aims

This study explores influence of damaged BBB and elevated P-gp on cerebral verapamil penetration after ischemia both in vivo and in vitro.

Methods

Middle cerebral artery occlusion (MCAO) induced ischemia/reperfusion (I/R) of rats, and Na₂S₂O₄ induced hypoxia/reoxygenation (H/R) damage of rat brain mirovessel endothelial cells (RBMECs) respectively, served as BBB breakdown model in vivo and in vitro. Evans-Blue (EB) extravagation and ¹²⁵I-albumin were used to quantify BBB dysfunction; UPLC–MS/MS analytical method was performed to determine accurately the concentration of verapamil in brain tissue and cell. Flow cytometry, immunohistochemistry and western blotting were applied to evaluate transport function and protein expression of P-gp.

Results

Overexpressed ICAM-1 and MMP-9 mediated BBB dysfunction after ischemia, which induced EB leakage and ¹²⁵I-albumin uptake increase. Enhanced accumulation of verapamil in brain tissue, but intracellular concentration reduced evidently after H/R injury. Transcellular transportation of verapamil elevated when P-gp function or expression was inhibited after H/R injury.

Conclusion

These data indicated that BBB penetration of verapamil under ischemia condition was not only depending on BBB breakdown, but also regulated by P-gp.     

Permeation of blood-borne IL15 across the blood-brain barrier and the effect of LPS

Interleukin15 (IL 15) is a proinflammatory cytokine with elevated concentrations in autoimmune diseases involving the periphery (e.g. rheumatoid arthritis) and CNS (e.g. multiple sclerosis). Its interactions with the blood-brain barrier (BBB) were studied in normal and lipopolysaccharide (LPS)-treated mice. ¹²⁵I-IL15 remained intact for at least 10 min after i.v. injection and reached CNS parenchyma with regional differences between brain and spinal cord. Both in vivo and in situ brain perfusion of ¹²⁵I-IL15 showed that its permeation of the BBB was non-saturable. LPS induced a significant increase of IL15 uptake by the brain and spinal cord, partly related to a higher general permeability of the BBB. The results suggest that the BBB is an interface for blood-borne IL15 to interact with the CNS in the basal state and during inflammation.     

Increased CNS uptake and enhanced antinociception of morphine-6-glucuronide in rats after inhibition of P-glycoprotein

Morphine-6-glucuronide (M6G) is a substrate of P-glycoprotein (P-gp), which forms an outward transporter at the blood-brain barrier. Inhibition of P-gp may therefore be expected to cause increased CNS uptake of M6G. We directly assessed the spinal concentrations of M6G and its antinociceptive effects in rats following pharmacological inhibition of P-gp. Spinal cord tissue concentrations of M6G were assessed by microdialysis with probes transversally implanted through the dorsal horns of the spinal cord at level L4. Ten rats received M6G intravenously (0.018 mg/kg loading dose plus 0.00115 mg/kg/min for an 8-h infusion), five of them together with PSC833 to inhibit P-gp (32-h infusion, starting 24 h before the addition of M6G). Antinociceptive effects were explored by means of formalin tests. After having obtained evidence for enhanced CNS uptake and antinociception of M6G in the presence of PSC833, additional behavioural experiments were performed in another 32 rats to assess the dose dependency of the antinociceptive effects of M6G either with or without PSC833 in comparison with both PSC833 alone and placebo. Inhibition of P-gp increased the M6G concentrations in the spinal cord approximately three-fold whereas the plasma concentrations were increased only by a factor of 1.4, which resulted in a more than doubled spinal cord/plasma concentration ratio (from 0.08 +/- 0.03 for M6G alone to 0.17 +/- 0.08 for M6G plus PSC833). Antinociceptive effects of M6G were significantly enhanced by inhibition of P-gp. Inhibition of P-gp alters the transport of M6G across the blood-brain barrier, resulting in enhanced spinal cord uptake and enhanced antinociception.     

Upregulation of the transport system for TNFalpha at the blood-brain barrier

The transport system for the cytokine tumor necrosis factor-alpha (TNFalpha) at the blood-brain barrier (BBB) enables an enhanced yet saturable entry of TNFalpha from blood to the CNS. This review focuses on the selective upregulation of the transport system for TNFalpha at the BBB that is specific for type of pathology, region, and time. The upregulation is reflected by increased CNS tissue uptake of radiolabeled TNFalpha after iv injection in mice and by inhibition of this increase with excess non-radiolabeled TNFalpha. (1) Spinal cord injury (SCI): upregulation of TNFalpha uptake after thoracic transection is seen in the delayed phase of BBB disruption at the lumbar spinal cord. Thoracic SCI by compression, however, has a longer lasting impact on TNFalpha transport that involves thoracic and lumbar spinal cord, in contrast to the upregulation confined to the lumbar region in lumbar SCI by compression. Regardless, the uptake of TNFalpha by spinal cord does not parallel BBB disruption as measured by the leakage of radiolabeled albumin. (2) Experimental autoimmune encephalomyelitis (EAE): the increase in the differential permeability to TNFalpha is seen in all CNS regions (brain and cervical, thoracic, and lumbar spinal cord) and has a distinct time course and reversibility. Exogenous TNFalpha has biphasic effects in modulating functional scores. The BBB, a dynamically regulated barrier, is actively involved in disease processes.     

Diurnal variation of leptin entry from blood to brain involving partial saturation of the transport system

The blood-brain barrier (BBB) regulates the amount of peripherally produced leptin reaching the brain. Knowing that the blood concentration of leptin has a circadian rhythm, we investigated whether the influx of leptin at the BBB followed the same pattern in three main sets of experiments. (a): The entry of 125I-leptin from blood to brain was measured in mice every 4 h, as indicated by the influx rate of 125I-leptin 1-10 min after an iv bolus injection. The blood concentration of endogenous leptin was measured at the same times. Blood leptin concentrations were higher at night and early morning (peak at 0800 h) and lower during the day (nadir at 1600 h). By contrast, the influx of 125I-leptin was fastest at 2000 h and slowest at 0400 h. Addition of unlabeled leptin (1 microg/mouse) significantly decreased the influx rate of 125I-leptin at all time points, indicating saturability of the transport system. The unlabeled leptin also abolished the diurnal variation of the influx of 125I-leptin. (b): The entry of 125I-leptin into spinal cord was faster than that into brain and showed a different diurnal pattern. The greatest influx occurred at 2400 h and the slowest at 0800 h. In spinal cord, unlike brain, unlabeled leptin (1 microg/mouse) neither inhibited the influx of 125I-leptin nor abolished the diurnal rhythm. (c): Higher concentrations of unlabeled leptin (5 microg/mouse) inhibited the uptake of 125I-leptin in spinal cord as well as in brain, but not in muscle. This experiment measured uptake 10 min after iv injection at 0600 h (beginning of the light cycle) and 1800 h (beginning of the dark cycle). Thus, influx of 125I-leptin into the CNS shows diurnal variation, indicating a circadian rhythm in the transport system at the BBB, saturation of the leptin transport system shows differences between the brain and spinal cord, and blood concentrations of leptin suggest that partial saturation of the transport system occurs at physiological concentrations of circulating leptin, contributing to the differing diurnal patterns in brain and spinal cord. Together, the results show that the BBB is actively involved in the neuroendocrine regulation of feeding behavior.     

In situ localization of P-glycoprotein (ABCB1) in human and rat brain.

Transport of several xenobiotics including pharmacological agents into or out of the central nervous system (CNS) involves the expression of ATP-dependent, membrane-bound efflux transport proteins such as P-glycoprotein (P-gp) at the blood-brain barrier (BBB). Previous studies have documented gene and protein expression of P-gp in brain microvessel endothelial cells. However, the exact localization of P-gp, particularly at the abluminal side of the BBB, remains controversial. In the present study we examined the cellular/subcellular distribution of P-gp in situ in rat and human brain tissues using immunogold cytochemistry at the electron microscope level. P-gp localizes to both the luminal and abluminal membranes of capillary endothelial cells as well as to adjacent pericytes and astrocytes. Subcellularly, P-gp is distributed along the nuclear envelope, in caveolae, cytoplasmic vesicles, Golgi complex, and rough endoplasmic reticulum (RER). These results provide evidence for the expression of P-gp in human and rodent brain capillary along their plasma membranes as well as at sites of protein synthesis, glycosylation, and membrane trafficking. In addition, its presence at the luminal and abluminal poles of the BBB, including pericytes and astrocyte plasma membranes, suggests that this glycoprotein may regulate drug transport processes in the entire CNS BBB at both the cellular and subcellular level.     

Nicotine and cotinine increases the brain penetration of saquinavir in rat

Endothelial tight junctions and efflux transporters of the blood-brain barrier (BBB) significantly limit brain accumulation of many drugs, including protease inhibitors such as saquinavir. The cholinergic agonist nicotine is one of the most commonly used drugs in the world and the incidence is even higher in the human immune deficiency virus population (～ 70%). We examined the ability of nicotine and its primary metabolite cotinine to modify brain uptake of saquinavir in rats. Both nicotine and cotinine at pharmacological concentrations matching those in smokers, increased brain saquinavir uptake by two fold. Co-perfusion with nicotinic receptor antagonists and passive permeability markers showed that the effect was not caused by receptor activation or BBB permeability disruption. Transport inhibition studies demonstrated that brain saquinavir uptake is limited by multiple efflux transporters, P-glycoprotein (P-gp), breast cancer resistance protein and multidrug resistance-associated protein. In situ perfusion and in vitro experiments using a classical P-gp substrate rhodamine 123 linked the effect of nicotine to inhibition of BBB P-gp transport. The effect was confirmed in vivo in chronic 14 day nicotine administration animals. These data suggest nicotine increases antiretroviral drug exposure to brain and may represent a significant in vivo drug-drug interaction at the BBB. Although this may slightly benefit CNS antiretroviral efficacy, it may also expose the brain to potential serious neurotoxicity.     

Uptake and Efflux of Quinacrine, a Candidate for the Treatment of Prion Diseases, at the Blood-Brain Barrier

1. A clinical trial of quinacrine in patients with Creutzfeldt-Jakob disease is now in progress. The permeability of drugs through the blood-brain barrier (BBB) is a determinant of their therapeutic efficacy for prion diseases. The mechanism of quinacrine transport across the BBB was investigated using mouse brain endothelial cells (MBEC4). 2. The permeability of quinacrine through MBEC4 cells was lower than that of sodium fluorescein, a BBB-impermeable marker. The basolateral-to-apical transport of quinacrine was greater than its apical-to-basolateral transport. In the presence of P-glycoprotein (P-gp) inhibitor, cyclosporine or verapamil, the apical-to-basolateral transport of quinacrine increased. The uptake of quinacrine by MBEC4 cells was enhanced in the presence of cyclosporine or verapamil. 3. Quinacrine uptake was highly concentrative, this event being carried out by a saturable and carrier-mediated system with an apparent Km of 52.1 microM. Quinacrine uptake was insensitive to Na+-depletion and changes in the membrane potential and sensitive to changes in pH. This uptake was decreased by tetraethylammonium and cimetidine, a substrate and an inhibitor of organic cation transporters, respectively. 4. These findings suggest that quinacrine transport at the BBB is mediated by the efflux system (P-gp) and the influx system (organic cation transporter-like machinery).     

Effect of cardiac arrest on brain weight and the permeability of the blood-brain and blood-spinal cord barrier to albumin and tumor necrosis factor-alpha

Time-dependent changes in brain and spinal cord were studied in mice in a cardiac arrest model. A transient decrease in body weight and a prolonged decrease in brain weight occurred after arrest whereas spinal cord weight was unchanged. The permeability of the blood-brain barrier (BBB) to I131-albumin and I131 tumor necrosis factor-alpha (TNF) showed maximal, non-significant increases on day 5 after cardiac arrest, but the permeability of the blood-spinal cord barrier (BSCB) to both materials was unchanged with time. We conclude that selective weight loss occurs in the brain after cardiac arrest with the integrity of the BBB and BSCB remaining intact to serum proteins and minimal alteration in the blood to CNS transport of TNF.     

Effects of hemorrhagic hypotension on tyrosine concentrations in rat spinal cord and plasma

Tyrosine is the precursor for catecholamine neurotransmitters. When catecholamine-containing neurons are physiologically active (as sympathoadrenal cells are in hypotension), tyrosine administration increases catecholamine synthesis and release. Since hypotension can alter plasma amino acid composition, we examined the effects of an acute hypotensive insult on tyrosine concentrations in plasma and spinal cord. Rats were cannulated and bled until the systolic blood pressure was 50 mmHg, or were kept normotensive for 1 h. Tyrosine and other large neutral amino acids (LNAA) known to compete with tyrosine for brain uptake were assayed in plasma and spinal cord. The rate at which intra-arterial [3H]tyrosine disappeared from the plasma was also estimated in hemorrhaged and control rats. In plasma of hemorrhaged animals, both the tyrosine concentration and the tyrosine/LNAA ratio was elevated; moreover, the disappearance of [3H]tyrosine was slowed. Tyrosine concentrations also increased in spinal cords of hemorrhaged-hypotensive rats when compared to normotensive controls. Changes in plasma amino acid patterns may thus influence spinal cord concentrations of amino acid precursors for neurotransmitters during the stress of hemorrhagic shock.     

Adverse Effect of Cyclosporin A on Barrier Functions of Cerebral Microvascular Endothelial Cells After Hypoxia-reoxygenation Damage In Vitro

Hypoxia and post-hypoxic reoxygenation induces disruption of the blood–brain barrier (BBB). Alterations of the BBB function after hypoxia/reoxygenation (H/R) injury remain unclear. Cyclosporin A (CsA), a potent immunosuppressant, induces neurotoxic effects by entering the brain, although the transport of CsA across the BBB is restricted by P-glycoprotein (P-gp), a multidrug efflux pump, and tight junctions of the brain capillary endothelial cells. The aim of this study was to evaluate whether the BBB after H/R damage is vulnerable to CsA-induced BBB dysfunction. We attempted to establish a pathophysiological BBB model with immortalized mouse brain capillary endothelial (MBEC4) cells. The effects of CsA on permeability and P-gp activity of the MBEC4 cells were then examined. Exposure to hypoxia for 4 h and reoxygenation for 1 h (H/R (4 h/1 h)) produced a significant decrease in P-gp function of MBEC4 cells, without changing cell viability and permeability for sodium fluorescein and Evan’s blue-albumin at 7 days after H/R (4 h/1 h). CsA-induced hyperpermeability and P-gp dysfunction in MBEC4 monolayers at 7 days after H/R (4 h/1 h) were exacerbated. The possibility that CsA penetrates the BBB with incomplete functions in the vicinity of cerebral infarcts to induce neurotoxicity has to be considered.     

Uptake and degradation of blood-borne insulin by the olfactory bulb.

Insulin found within the brain is derived from the blood and can affect various central nervous system (CNS) functions. The olfactory bulb contains one of the highest concentrations of insulin and insulin receptors within the CNS. To determine the mechanism underlying this high concentration of insulin, we used radioactively iodinated insulin to compare the blood to tissue transport rates and tissue degradation rates for the olfactory bulb, whole brain and spinal cord. We found that the olfactory bulb had both the highest transport rate across the blood-brain barrier (BBB) and the highest rate of degradation. Because a higher degradation rate would decrease, not increase, tissue concentrations of insulin, BBB transport may be the primary mechanism by which high concentrations of insulin are maintained within the olfactory bulb. This illustrates an adaptive aspect of the BBB in its regulation of the exchange of information molecules between the blood and the CNS.     

Induction and clinical scoring of chronic-relapsing experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by demyelination and glial scaring in areas disseminated in the brain and spinal cord. These lesions alter nerve conduction and induce the disabling neurological deficits that vary with the location of the demyelinated plaques in the CNS (e.g. paraparesis, paralysis, blindness, incontinence). Experimental autoimmune encephalomyelitis (EAE) is a model for MS. EAE was first induced accidentally in humans during vaccination against rabies, using viruses grown on rabbit spinal cords. Residues of spinal injected with the inactivated virus induced the CNS disease. Following these observations, a first model of EAE was described in non-human primates immunized with a CNS homogenate by Rivers and Schwenther in 1935. EAE has since been generated in a variety of species and can follow different courses depending on the species/strain and immunizing antigen used. For example, immunizing Lewis rats with myelin basic protein in emulsion with adjuvant induces an acute model of EAE, while the same antigen induces a chronic disease in guinea pigs. The EAE model described here is induced by immunizing DA rats against DA rat spinal cord in emulsion in complete Freund's adjuvant. Rats develop an ascending flaccid paralysis within 7-14 days post-immunization. Clinical signs follow a relapsing-remitting course over several weeks. Pathology shows large immune infiltrates in the CNS and demyelination plaques. Special considerations for taking care for animals with EAE are described at the end of the video.     

Centrally Administered Pertussis Toxin Inhibits Microglia Migration to the Spinal Cord and Prevents Dissemination of Disease in an EAE Mouse Model

Background

Experimental autoimmune encephalomyelitis (EAE) models are important vehicles for studying the effect of infectious elements such as Pertussis toxin (PTx) on disease processes related to acute demyelinating encephalomyelitis (ADEM) or multiple sclerosis (MS). PTx has pleotropic effects on the immune system. This study was designed to investigate the effects of PTx administered intracerebroventricularly (icv) in preventing downstream immune cell infiltration and demyelination of the spinal cord.

Methods and Findings

EAE was induced in C57BL/6 mice with MOG_35–55. PTx icv at seven days post MOG immunization resulted in mitigation of clinical motor symptoms, minimal T cell infiltration, and the marked absence of axonal loss and demyelination of the spinal cord. Integrity of the blood brain barrier was compromised in the brain whereas spinal cord BBB integrity remained intact. PTx icv markedly increased microglia numbers in the brain preventing their migration to the spinal cord. An in vitro transwell study demonstrated that PTx inhibited migration of microglia.

Conclusion

Centrally administered PTx abrogated migration of microglia in EAE mice, limiting the inflammatory cytokine milieu to the brain and prevented dissemination of demyelination. The effects of PTx icv warrants further investigation and provides an attractive template for further study regarding the pleotropic effects of infectious elements such as PTx in the pathogenesis of autoimmune disorders.     

P-Glycoprotein Mediated Efflux Limits the Transport of the Novel Anti-Parkinson's Disease Candidate Drug FLZ across the Physiological and PD Pathological In Vitro BBB Models

FLZ, a novel anti-Parkinson''s disease (PD) candidate drug, has shown poor blood-brain barrier (BBB) penetration based on the pharmacokinetic study using rat brain. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two important transporters obstructing substrates entry into the CNS as well as in relation to PD neuropathology. However, it is unclear whether P-gp and BCRP are involved in low BBB permeability of FLZ and what the differences of FLZ brain penetration are between normal and Parkinson''s conditions. For this purpose, in vitro BBB models mimicking physiological and PD pathological-related BBB properties were constructed by C6 astroglial cells co-cultured with primary normal or PD rat cerebral microvessel endothelial cells (rCMECs) and in vitro permeability experiments of FLZ were carried out. High transepithelial electrical resistance (TEER) and low permeability for sodium fluorescein (NaF) confirmed the BBB functionality of the two models. Significantly greater expressions of P-gp and BCRP were detected in PD rCMECs associated with the lower in vitro BBB permeability of FLZ in pathological BBB model compared with physiological model. In transport studies only P-gp blocker effectively inhibited the efflux of FLZ, which was consistent with the in vivo permeability data. This result was also confirmed by ATPase assays, suggesting FLZ is a substrate for P-gp but not BCRP. The present study first established in vitro BBB models reproducing PD-related changes of BBB functions in vivo and demonstrated that poor brain penetration of FLZ and low BBB permeability were due to the P-gp transport.     

Changes in dipole membrane potential at the mouse blood–brain barrier enhance the transport of 99mTechnetium Sestamibi more than inhibiting Abcb1, Abcc1, or Abcg2

Cationic ^99mTc-agents like ^99mTc-hexakis-2-methoxyisobutyl isonitrile (^99mTc-MIBI) cannot be used for brain imaging because they do not enter the brain as readily as some uncharged ^99mTc-compounds. The mechanism by which cationic ^99mTc-agents are transported across the blood–brain barrier (BBB) remains unclear. We explored ^99mTc-MIBI transport by in situ mouse brain perfusion to determine the influence of BBB features like the ATP-binding cassette transporters (Abcb1/P-glycoprotein (P-gp), Abcc1/Mrp1, and Abcg2/Bcrp), organic cation transporters (Slc22a1-3/Oct1-3), the transmembrane potential and the dipole membrane potential. P-gp reduced ^99mTc-MIBI transport across the BBB of P-gp-deficient mice 2.2-fold, as confirmed by PSC833 and GF120918 inhibition. Paradoxically verapamil decreased its transport '0.6-fold'. Reducing the BBB dipole membrane potential with tetraphenylborate or phloretin increased ^99mTc-MIBI transport about 12- and 20-fold, respectively. Guanidine, diphenhydramine, and carnitine significantly decreased ^99mTc-MIBI transport, but tetraethylammonium did not. ^99mTc-MIBI transport at the BBB is restricted by P-gp but not by Mrp1 or Bcrp. Some organic cations reduced the influx of ^99mTc-MIBI into the brain independently of Oct1, 2 and 3, but this could be due to their effect on another cation transporter. The membrane dipole potential of the luminal BBB membrane appeared to be the main factor restricting ^99mTc-MIBI permeability.     

Fructose Levels Are Markedly Elevated in Cerebrospinal Fluid Compared to Plasma in Pregnant Women

Background

Fructose, unlike glucose, promotes feeding behavior in rodents and its ingestion exerts differential effects in the human brain. However, plasma fructose is typically 1/1000th of glucose levels and it is unclear to what extent fructose crosses the blood-brain barrier. We investigated whether local endogenous central nervous system (CNS) fructose production from glucose via the polyol pathway (glucose→sorbitol→fructose) contributes to brain exposure to fructose.

Methods

In this observational study, fasting glucose, sorbitol and fructose concentrations were measured using gas-chromatography-liquid mass spectroscopy in cerebrospinal fluid (CSF), maternal plasma, and venous cord blood collected from 25 pregnant women (6 lean, 10 overweight/obese, and 9 T2DM/gestational DM) undergoing spinal anesthesia and elective cesarean section.

Results

As expected, CSF glucose was ~60% of plasma glucose levels. In contrast, fructose was nearly 20-fold higher in CSF than in plasma (p < 0.001), and CSF sorbitol was ~9-times higher than plasma levels (p < 0.001). Moreover, CSF fructose correlated positively with CSF glucose (ρ 0.45, p = 0.02) and sorbitol levels (ρ 0.75, p < 0.001). Cord blood sorbitol was also ~7-fold higher than maternal plasma sorbitol levels (p = 0.001). There were no differences in plasma, CSF, and cord blood glucose, fructose, or sorbitol levels between groups.

Conclusions

These data raise the possibility that fructose may be produced endogenously in the human brain and that the effects of fructose in the human brain and placenta may extend beyond its dietary consumption.     

Dexamethasone Regulation of P-Glycoprotein Activity in an Immortalized Rat Brain Endothelial Cell Line, GPNT

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from the blood to the extracellular fluid environment of the brain. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain capillary endothelial cells, thus forming a functional barrier to lipid-soluble drugs, notably, antitumor agents. It is of interest to develop an in vitro BBB model that stably expresses P-gp to investigate the mechanisms of regulation in expression and activity. The rat brain endothelial cell line, GPNT, was derived from a previously characterized rat brain endothelial cell line. A strong expression of P-gp was found in GPNT monocultures, whereas the multidrug resistance-associated pump Mrp1 was not expressed. The transendothelial permeability coefficient of the P-gp substrate vincristine across GPNT monolayers was close to the permeability coefficient of bovine brain endothelial cells cocultured with astrocytes, a previously documented in vitro BBB model. Furthermore, the P-gp blocker cyclosporin A induced a large increase in apical to basal permeability of vincristine. Thus, P-gp is highly functional in GPNT cells. A 1-h treatment of GPNT cells with dexamethasone resulted in decreased uptake of vincristine without any increase in P-gp expression. This effect could be mimicked by protein kinase C (PKC) activation and prevented by PKC inhibition, strongly suggesting that activation of P-gp function may involve a PKC-dependent pathway. These results document the GPNT cell line as a valuable in vitro model for studying drug transport and P-gp function at the BBB and suggest that activation of P-gp activity at the BBB might be considered in chemotherapeutic treatment of cancer patients.     

Sufficient virus-neutralizing antibody in the central nerve system improves the survival of rabid rats

Background

Rabies is known to be lethal in human. Treatment with passive immunity for the rabies is effective only when the patients have not shown the central nerve system (CNS) signs. The blood–brain barrier (BBB) is a complex functional barrier that may compromise the therapeutic development in neurological diseases. The goal of this study is to determine the change of BBB integrity and to assess the therapeutic possibility of enhancing BBB permeability combined with passive immunity in the late stage of rabies virus infection.

Methods

The integrity of BBB permeability in rats was measured by quantitative ELISA for total IgG and albumin levels in the cerebrospinal fluid (CSF) and by exogenously applying Evans blue as a tracer. Western blotting of occludin and ZO-1, two tight junction proteins, was used to assess the molecular change of BBB structure.The breakdown of BBB with hypertonic arabinose, recombinant tumor necrosis factor-alpha (rTNF-γ), and focused ultrasound (FUS) were used to compare the extent of BBB disruption with rabies virus infection. Specific humoral immunity was analyzed by immunofluorescent assay and rapid fluorescent focus inhibition test. Virus-neutralizing monoclonal antibody (mAb) 8-10E was administered to rats with hypertonic breakdown of BBB as a passive immunotherapy to prevent the death from rabies.

Results

The BBB permeability was altered on day 7 post-infection. Increased BBB permeability induced by rabies virus infection was observed primarily in the cerebellum and spinal cord. Occludin was significantly decreased in both the cerebral cortex and cerebellum. The rabies virus-specific antibody was not strongly elicited even in the presence of clinical signs. Disruption of BBB had no direct association with the lethal outcome of rabies. Passive immunotherapy with virus-neutralizing mAb 8-10E with the hypertonic breakdown of BBB prolonged the survival of rabies virus-infected rats.

Conclusions

We demonstrated that the BBB permeability was altered in a rat model with rabies virus inoculation. Delivery of neutralizing mAb to the infected site in brain combined with effective breakdown of BBB could be an aggressive but feasible therapeutic mode in rabies when the CNS infection has been established.     

Neuregulin-1-beta1 enters brain and spinal cord by receptor-mediated transport

Proteins of the neuregulin (NRG) family play important regulatory roles in neuronal survival and synaptic activity. NRG-1-beta1 has particular potential as a therapeutic agent because it enhances myelination of neurites in spinal cord explants. In this study, we determined the permeation of NRG-1-beta1 across the blood-brain and blood-spinal cord barriers (BBB and BSCB respectively). Intact radioactively labeled NRG-1-beta1 had a saturable and relatively rapid influx rate from blood to the CNS in mice. Capillary depletion studies showed that NRG-1-beta1 entered the parenchyma of the brain and spinal cord rather than being trapped in the capillaries that compose the BBB. The possible mechanism of receptor-mediated transport was shown by the ability of antibodies to erbB3 and erbB4 receptors to inhibit the influx. Lipophilicity, less important for such saturable transport mechanisms, was measured by the octanol : buffer partition coefficient and found to be low. The results indicate that NRG-1-beta1 enters spinal cord and brain by a saturable receptor-mediated mechanism, which provides the opportunity for possible therapeutic manipulation at the BBB level.