Cerebrovascular protection as a possible mechanism for the protective effects of NXY-059 in preclinical models: An in vitro study

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Cerebrovascular protection as a possible mechanism for the protective effects of NXY-059 in preclinical models: An in vitro study Maxime Culot a,b,⁎, Caroline Mysiorek a,b , Mila Renftel c , Benoit D. Roussel d , Yannick Hommet d , Denis Vivien d , Roméo Cecchelli a,b , Laurence Fenart a,b , Vincent Berezowski a,b , Marie-Pierre Dehouck a,b , Stefan Lundquist c a

Univ Lille-Nord de France, F59000 Lille, France UArtois, BBB laboratory, EA 2465, F62300 Lens, France c Department of Drug Metabolism and Pharmacokinetics, AstraZeneca R&D Södertälje, S-151 85 Södertälje, Sweden d INSERM U919, Serine Proteases and Pathophysiology of the Neurovascular Unit, UMR-CNRS 6232 CINAPS, Cyceron, Université de Caen Basse-Normandie, Caen, F-14074, France b

A R T I C LE I N FO

AB S T R A C T

Article history:

NXY-059, a polar compound with limited transport across the blood-brain barrier, has

Accepted 14 July 2009

demonstrated neuroprotection in several animal models of acute ischemic stroke but

Available online 23 July 2009

failed to confirm clinical benefit in the second phase III trial (SAINT-II). To improve the understanding of the mechanisms responsible for its neuroprotective action in preclinical

Keywords:

models a series of experiments was carried out in an in vitro blood-brain barrier (BBB)

Blood-brain barrier

model. A clinically attainable concentration of 250 μmol/L of NXY-059 administered at the

Endothelium

onset or up to 4 h after oxygen glucose deprivation (OGD) produced a significant

Ischemia

reduction in the increased BBB permeability caused by OGD. Furthermore, OGD produced

Neuroprotection

a huge influx of tissue plasminogen activator across the BBB, which was substantially

NXY-059

reduced by NXY-059. This study suggests that the neuroprotective effects of NXY-059

Stroke

preclinically, may at least in part be attributed to its ability to restore functionality of the brain endothelium. © 2009 Elsevier B.V. All rights reserved.

1.

Introduction

By the year 2020, stroke is expected to be the fourth leading disease burden worldwide (Michaud et al., 2001). For many years, researchers have hoped that agents which have been shown to be neuroprotective in vitro or in animal models of

ischemic stroke would also prove successful in stroke patients. The disappointing clinical results so far, reflect the lack of understanding of post ischemic brain injury and it has been suggested that by expanding the focus to include the neurovascular unit (endothelium, neurons, astrocytes, oligodendrocytes, extracellular matrix, basal

⁎ Corresponding author. Laboratoire de physiopathologie de la Barrière Hémato-Encéphalique, Université Lille-Nord de France, EA2465, IMPRT:IFR114, Faculté des Sciences Jean Perrin, rue Jean Souvraz, F62300 Lens, France. E-mail address: [email protected] (M. Culot). Abbreviations: BBB, blood-brain barrier; BSA, bovine serum albumin; ECs, endothelial cells; GCs, glial cells; OGD, oxygen glucose deprivation; PBS, phosphate-buffered saline; Pe, endothelial permeability coefficient; tPA, tissue plasminogen activator 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.07.035

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lamina, pericytes and circulating blood elements) a more integrative answer to brain damage after ischemic injury might be obtained (del Zoppo, 2006). The blood-brain barrier (BBB), located at the level of the cerebral capillaries is a unique feature of the cerebrovascular system which regulates molecular exchange between the brain and systemic circulation as well as transmigration of circulating blood cells. This dynamic interface can undergo rapid transformation and become actively prothrombotic and proinflammatory in response to various mediators (Cecchelli et al., 2007). Deprivation of blood supply may lead to dysregulation of transport of vital nutrients and changes in the overall permeability of the BBB, resulting in oedema formation and matrix degradation with associated hemorrhagic transformation. So although neuronal cells have long been thought to be prime targets in ischemia, alterations that affect the cerebrovascular–parenchymal interface are of key importance for the initiation of the cascade of events which ultimately results in ischemic tissue. The BBB may therefore constitute an important therapeutic target for protecting neurons and glial cells, subsequent to an ischemic insult (Fagan et al., 2004). NXY-059 is a free radical-trapping agent that reduces infarct size and preserves brain function in several animal models of acute ischemic stroke but failed to confirm benefit in acute ischemic stroke patients in the second of two pivotal phase III clinical trials (SAINT-II) (Diener et al., 2008). Despite the disappointing outcome of the SAINT-II study, it is still important to improve the understanding of the mechanisms responsible for the neuroprotective action of NXY-059 in preclinical models. NXY-059 is a highly polar compound and has previously been shown to have limited permeability across the BBB under physiological conditions in vitro (Dehouck et al., 2002) as well as in transient ischemic models (Kuroda et al., 1999). However, the blood-brain barrier was found to be severely compromised in animal models of permanent ischemia as well as in an in vitro model of the blood-brain barrier mimicking permanent ischemia (Brillault et al., 2002; Dehouck et al., 2002; Green et al., 2006; Plateel et al., 1995, 1997). In an attempt to address the molecular events that underlie the neuroprotective effect of NXY-059, possible mechanisms of action related to the cerebral capillary endothelium and glial cells were investigated.

2.

Results

2.1. Effect of NXY-059 on bovine brain capillary endothelia in severe ischemia An in vitro model of the blood-brain barrier was used to investigate the effects of NXY-059 on brain endothelial and glial cells in normoxic and ischemic conditions, (Fig. 1a). The model is well characterized and possesses many of the characteristic properties of the in vivo BBB (Cecchelli et al., 1999, 2007; Dehouck et al., 1992; Lundquist et al., 2002). It has previously been used to study BBB function in hypoxic and ischemic conditions (Brillault et al., 2002; Dehouck et al., 2002; Plateel et al., 1995). The tightness of the in vitro BBB in all experiments throughout this study was monitored through

Fig. 1 – NXY-059 reduces the ischemia-induced increase of sucrose permeability in brain capillary endothelial cells. (a) Schematic representation of the in vitro BBB model. (b) 250 μmol//L NXY-059 was applied in the luminal compartment of the coculture at the onset of the 4 h OGD insult or (c) the whole coculture was pretreated for 24 h with 20 μmol/L NXY-059 before the onset of the 4 h OGD. Data are expressed as mean ± SEM percentage of [14C]-sucrose transport. The 100% control value represents a Pe of 0.56 × 10− 3 (a) or (b) 0.39 × 10− 3 cm/min obtained after 4 h in normoxic conditions. **p < 0.01 and ***p < 0.001 versus OGD without treatment by a Dunnet's post-hoc test after a significant ANOVA (p < 0.001; n = 3 independent EC monolayers/treatment).

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the use of sucrose as an integrity marker. Sucrose Pe values below 1 × 10− 3 cm/min have previously been determined to correlate with well-developed tight junctions in the model (Culot et al., 2008; Lundquist et al., 2002), and were in all normoxic experiments found to be in the range of 0.27 to 0.56 × 10− 3 cm/min. To investigate the effect of NXY-059 on a dysfunctional BBB caused by ischemic conditions, the in vivo insult was simulated in vitro by depriving cells of oxygen and glucose for 4 h. In these experiments, NXY-059 was placed in the luminal compartment (i.e. the blood-side) at a clinically attainable concentration of 250 μmol/L. As shown in Fig. 1b, OGD caused a 9 fold increase in sucrose permeability which was reduced by 50% in the presence of NXY-059 but did not result in a complete restoration of in vitro BBB functionality. Furthermore, the effect of pretreatment of the cells with NXY-059 was investigated. It was found that a 24 h pretreatment of the coculture (i.e. endothelial and glial cells) with 20 μmol/L NXY-059, (a concentration which is consistent with the extracellular brain concentration estimated from in vivo data (Green et al., 2006)) resulted in a similar reduction of BBB permeability as achieved by exposing only the brain endothelium to 250 μmol/L NXY-059 from the onset of OGD (Fig. 1c). However, in neither case were there complete restorations of the properties of the in vitro BBB. It has previously been reported that hypoxic and ischemic conditions induce secretion of factors from glial cells that compromise the integrity of the in vitro BBB with ensuing increase in the abluminal concentration of both large molecules such as albumin and tPA as well as smaller molecules such as sucrose, inuline and NXY-059 (Benchenane et al., 2005b; Brillault et al., 2002; Dehouck et al., 2002). Therefore, the mechanism of action of NXY-059 could be related to inhibition of deleterious secretion of factors from glial cells and/or due to an effect exerted on the brain endothelium. In an attempt to discriminate the cell population primarily associated with the protective action of NXY-059 (endothelial cells or glial cells), cell populations were either untreated or subjected to 24 h pretreatments with NXY-059 followed by assessment of permeability across the brain endothelium (Fig. 2a). By combining pretreated brain endothelium with untreated glial cells or vice versa followed by assessment of sucrose permeability across the pretreated or untreated endothelial cell monolayer, it could be shown that only pretreatment of endothelial cells with NXY-059 results in a reduction of the increased permeability caused by OGD. However, similarly to the previous experiments there is no complete restoration of the functionality of the in vitro BBB but the results suggest that the protective effect of NXY-059 is mediated by mechanisms primarily related to the cerebral endothelium (Fig. 2b). In the clinical studies (SAINT-I and SAINT-II) patients were treated within 6 h from the onset of stroke with doses of NXY059 comparable with those generating neuroprotective effects in animal models (Lapchak et al., 2004; Marshall et al., 2003). This made it relevant to investigate whether NXY-059 was also protective in vitro if administered at some later time points within this treatment window (Fig. 3a). Exposing brain endothelium and glial cells to 6 h of OGD, produced a huge increase in the permeability of the BBB (Fig. 3b). Administering NXY-059 either at the onset, 2.5 or 4 h after the insult

Fig. 2 – The protective effect of NXY-059 pretreatment is due to its effect on brain capillary endothelial cells. (a) Schematic diagram of the experimental protocol: EC and glial cells were pretreated with 20 μmol/L NXY-059, 24 h before the experiment. At the onset of the experiment, EC inserts from NXY-059 pretreated coculture were place in wells containing untreated glial cells and the EC inserts from untreated coculture were place in the wells of the pretreated coculture (i.e. containing NXY-059 pretreated glial cells). Then the medium of coculture was replaced by OGD medium and cells were incubated for 4 h in ischemic conditions. (b) Mean ± SEM percentage of [14C]-sucrose transport. The 100% control value represents a Pe of 0.28 × 10− 3 cm/min obtained after 4 h in normoxic conditions. ***p < 0.001 versus OGD without treatment by a Dunnet's post-hoc test after a significant ANOVA (p < 0.001; n = 3 independent EC monolayers/treatment).

generated in all cases statistically significant reductions of the increase in sucrose permeability caused by OGD but no complete restoration of BBB functionality was observed. These

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results are in agreement with in vivo results presented by Kuroda et al. (1999), which showed that administration of NXY-059 3 to 6 h after the beginning of the reperfusion in a rat transient MCAO model reduced infarct volume. The administration of NXY-059 at 4 h after the onset of OGD resulted in a similar protection as obtained when NXY-059 was present during the whole period of OGD (Fig. 3b). However, this effect was found to be concentration dependent and occurred only at concentrations of NXY-059 which were not below 250 μmol/L (Fig. 3c). To confirm that the effect of late administration of NXY059 was a result of its direct effect on the brain endothelium, experiments described in Fig. 4a were carried out. Endothelial cells were first submitted to ischemic conditions for 4 h and then for 2 additional hours of ischemia without glial cells. The increase in the BBB permeability to sucrose caused by

147

ischemic conditions, has previously been suggested to be related to a direct involvement of glial cells (Brillault et al., 2002). This deleterious role of glial cells in OGD was confirmed in these experiments since no increase in sucrose permeability was observed in OGD without “glial input”. In contrast, the presence of glial cells during the first 4 h of OGD caused a significant increase in the BBB permeability of sucrose. A combination of 4 h of OGD with glial cells present, followed by 2 h luminal treatment of endothelial cells with 250 μmol/L NXY-059 during the second phase of the experiment (when glial cells are not present) resulted in a 2-fold decrease in the OGD-induced increase in BBB permeability but did not result in a complete normalisation of BBB function. These results clearly indicate that the cerebrovascular protective effect exerted by NXY-059 is most likely caused by a direct action on the brain endothelium. Reported data, in vivo (Cipolla et al., 2004; Kaur et al., 2006) and in vitro (Brillault et al., 2002), indicate that dysregulated transcytosis across the brain endothelium may be a feature of the compromised BBB in the early stages of ischemia but the underlying mechanisms are still unclear. On comparing the effects on the BBB caused by an osmotic shock with those generated by an ischemic insult, immunofluorescent microscopy revealed distinct differences at the level of the tight junctions. An osmotic shock caused by 30 min of mannitol treatment (1.4 mol/L), had a clear effect on occludin, one of the major tight junction proteins (Fig. 5a). No visible changes in the immunostaining pattern of this protein could be detected in ischemic conditions with or without NXY-059 present (Fig. 5b). However, this does not rule out the possibility that other tight junction proteins are compromised following an ischemic insult.

Fig. 3 – NXY-059 at 250 μM is as protective when added up to 4 h after the insult as when present during the whole period of OGD. (a) Schematic diagram of the experimental protocol: NXY-059 was added in the luminal compartment of the coculture at the onset of OGD or 2.5 and 4 h after the onset of the OGD insult. The BBB permeability to sucrose was evaluated 6 h after the onset of the experiment. (b) 250 μmol// L NXY-059 was added in the luminal compartment of the coculture at the onset of OGD or 2.5 and 4 h after the onset of the 6 h OGD insult. Data are expressed as mean ± SEM percentage of [14C]-sucrose transport. The 100% control value represents a Pe of 0.27 × 10− 3 cm/min obtained after 6 h in normoxic conditions. **p < 0.01 versus OGD without treatment by a Dunnet's post-hoc test after a significant ANOVA (p < 0.001; n = 3 independent EC monolayers/ treatment). n.s.: no statistical difference was noticed in the magnitude of the effect exerted by NXY-059 when added at different time after the onset OGD. (c) NXY-059 at 2.5, 25 or 250 μmol/L was added in the luminal compartment of the coculture 4 h after the onset of the 6 h OGD insult. Data are presented as mean± SEM percentage of [14C]-sucrose transport. The 100% control value represents a Pe of 0.49 × 10− 3 cm/min obtained after 6 h in normoxic conditions. *p< 0.05 versus OGD without treatment by a Dunnet's post-hoc test after a significant ANOVA (p<0.001; n = 3 independent EC monolayers/treatment).

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across the BBB (Benchenane et al., 2005b). tPA has been shown to promote both desirable (thrombolytic) and undesirable (neurotoxic) effects in stroke (Benchenane et al., 2004). A compromised BBB, caused by an ischemic insult, could result in the brain entry of relatively high amounts of tPA derived from the circulation that may potentiate deleterious effects on neuronal and glial cells (Nicole et al., 2001). To investigate the effect of NXY-059 on transport of tPA to the abluminal side of the BBB, 20 mg/L tPA was added in the luminal compartment after 3 h of normoxia or OGD, with or without NXY-059 present. After 2 h, the tPA activity in the abluminal media was analysed by a fluorogenic substrate assay. The integrity of the BBB was monitored by parallel measurements of sucrose permeability. The results of these experiments are summarised in Fig. 6. Firstly there was no direct inhibition of 2 mg/L or 20 mg/L tPA activity following 3 h incubation at 37 °C in serum-free medium with or without 250 μM NXY-059 present (data not shown). The results demonstrated that NXY-059 has no effect on the transport of tPA across the BBB in normoxic conditions (Fig. 6a). In contrast, ischemic conditions resulted in a much greater influx of tPA from the luminal to the abluminal compartment which was greatly reduced in the presence of NXY-059 (250 μmol/L). The corresponding measurements of sucrose transport confirmed previous results, showing an increase in sucrose BBB permeability following the ischemic insult, which was significantly reduced by 250 μmol/L NXY-059 (Fig. 6b).

3. Fig. 4 – The protective effect of the delayed administration of NXY-059 is due to its effect on brain capillary endothelial cells. (a) Schematic diagram of the experimental protocol: At the onset of the experiment ECs were first submitted to 4 h of either normoxia or ischemia with or without glial cells present. Then at 4 h, cell culture inserts were transferred into new wells without glial cells and submitted to another 2 h of either normoxia or ischemia in the presence or absence of 250 μmol/L of NXY-059 in the luminal compartment. (b) Mean ± SEM percentage of [14C]-sucrose transport. The 100% control value represents a Pe of 0.47 × 10− 3 cm/min obtained after 4 h in normoxic conditions with glial cells and 2 additional hours of normoxia without glial cells. **p < 0.01 versus OGD without NXY-059 present by a Dunnet's post-hoc test after a significant ANOVA (p < 0.001; n = 3 independent EC monolayers/treatment).

2.2. Effect of NXY-059 on the transport of tPA across the blood-brain barrier It has previously been reported that tPA is transported across the BBB independently of its proteolytic activity in vitro and in vivo and uses receptor-mediated transport via LRP (lowdensity lipoprotein receptor-related protein) in normoxic conditions (Benchenane et al., 2005a). However, in OGD, this receptor-mediated transport is altered to a non-specific and non-saturable process, which results in a huge influx of tPA

Discussion

The preclinical animal data for NXY-059 are convincing, but were not predictive since no benefit of NXY-059 could be established in the second Phase III clinical trial (SAINT-II). Since the NXY-059 is known to have limited BBB permeability (Dehouck et al., 2002; Kuroda et al., 1999), it has been suggested that this compound was not directly rescuing neurons from free radical injury, but rather exerts effects at the level of the cerebral endothelium (Kuroda et al., 1999; Maples et al., 2004). The evaluation of the effect of NXY-059 on BBB functions in ischemic conditions, using an in vitro model of the BBB demonstrates a clear protection by NXY-059 against the OGD-induced increase in BBB permeability, even when added 4 h after the onset of the insult (Fig. 3). These findings are consistent with the preclinical evaluation of NXY-059 (Kuroda et al., 1999; Sydserff et al., 2002). Furthermore, the effects of pretreatment as well as late administration of NXY059, clearly demonstrate that at least part of its protective effect is caused by a direct effect on the brain endothelium (Figs. 2–4). Under physiological conditions, complex tight junctions and low transcytotic activity at the level of the BBB makes the brain practically inaccessible for hydrophilic substances unless they are transferred by transport pathways across the endothelium. The tightness of the in vitro BBB in all experiments throughout this study was monitored through the use of sucrose as an integrity marker. The low permeabilities to sucrose, below 1 × 10− 3 cm/min obtained in normoxic conditions have previously been determined to correlate with welldeveloped tight junctions in the model (Culot et al., 2008;

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Fig. 5 – Immunostaining of endothelial cells with antibody raised against occludin a protein of tight junctions, treated or not treated with NXY-059. (a) Positive control for breakdown of tight junctions was evidenced after 30 min of mannitol treatment. (b) No modification of the tight junction protein occludin could be observed after 4 h of ischemia. Scale bar, 25 μm.

Lundquist et al., 2002). Interestingly, NXY-059 improved the properties of the compromised BBB in all experiments but was never capable of completely restoring the functionality of the in vitro BBB in terms of sucrose permeability, and it is tempting to speculate whether this could be linked to the disappointing outcome of the clinical trials. The observed continuous reticular localisation of the tight junction protein, occludin at the cell borders even following 4 h of OGD, suggests that cells were sealed together in both normoxic and ischemic conditions but does not rule out that other tight junction proteins might be compromised following the insult. However, the increase in BBB permeability of the marker molecule [14C]-sucrose under OGD clearly revealed a loss of barrier function in line with our previous observations in vitro (Brillault et al., 2002). These results are consistent with an early BBB dysfunction and suggest that OGD may lead to altered transport properties of the brain endothelium, which are not necessarily directly linked to the tight junctions. For instance, Cipolla et al. (2004) has reported enhanced pinocytic vesicle formation following transient ischemia and elevated intravascular pressure. Further studies are clearly needed to address the effects an ischemic insult has on the integrity and transport properties of the BBB. To date, the clot-busting drug tissue-type plasminogen activator remain the only FDA-approved therapy for acute stroke. Despite uncontroversial benefit, tPA has been shown to promote both desirable (thrombolytic) and undesirable (neurotoxic) effects in stroke (Benchenane et al., 2004). One of the exciting findings of the SAINT-I study (although not confirmed

in SAINT-II), was that patients receiving both tPA and NXY-059 had a significantly lower risk of hemorrhage (Lees et al., 2006). This prompted the investigation of the possible effects NXY059 might have on tPA. The study demonstrated, that although there was no interaction between the two molecules which lead to the inactivation of tPA, the ischemic conditions caused a huge flux of tPA across the BBB to the abluminal (brain) compartment which was dramatically reduced by NXY-059 at 250 μM (Fig. 6a). These data are interesting, since they indicate that, NXY-059 may limit tPA distribution into brain parenchyma. This may result in a protective effect on neuronal and glial cells, which is consistent with the reported benefits of combination therapy with tPA and NXY-059 preclinically (Lapchak et al., 2002a,b, 2004). The present study demonstrates the ability of NXY-059 to partially restore functionality of brain endothelium by reducing a pathologically upregulated BBB permeability. Thus, this mechanism of action may lead to an improved brain homeostasis by inhibiting the deleterious cascades of events that are initiated at the level of the cerebral vasculature following an ischemic insult. The work of Siesjö et al. suggested a neuroprotective action of NXY-059 in a transient model of ischemia (Yoshimoto et al., 2002). However, a major concern with the hypothesis of a direct neuroprotective action of NXY059 was that the same group under the same experimental conditions previously had shown that there were negligible amounts of NXY-059 present in the brain (Kuroda et al., 1999). Thus, the neuroprotective effect may emanate from restored functionality of the brain endothelium rather than direct

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reported by Hainsworth et al. (2008), however, demonstrating that a concentration of 300 μmol/L of NXY-059 failed to protect neuron-like cells from free radical-induced damage, add further support to the hypothesis that NXY-059 in preclinical models acts at the cerebrovascular–parenchymal interface rather than on a neuronal level. NXY-059, was the first neuroprotective compound developed, which was evaluated according to the STAIR criteria before entering Phase III clinical trials (Green and Ashwood, 2005). The fact that the clinical trials despite these efforts failed to demonstrate benefit emphasises that preclinical animal models of stroke need to be improved to be able to predict human pathology and it may be appropriate to conclude with the following statement, appearing in a recent review of the reasons behind failures in stroke research by Green and Shuaib (2006): “Animal models almost invariably use young healthy animals, whereas stoke patients are usually elderly, with a variety of other clinical problems, such as hypertension, myocardial infarction and diabetes, and generally none of these problems is included in the animal model”.

4.

Experimental procedures

All experiments were performed in the framework of the French legislation that governs animal experimentation (International permission number: A-62-498-5).

Fig. 6 – Effect of NXY-059 on the transport of tPA across the BBB. 20 μg/mL tPA was added in the luminal compartment with or without NXY-059 present. (a) Following 3 h in normoxic or OGD conditions, tPA activity in abluminal media was analysed by fluorogenic substrate assay. Data are expressed as mean ± SEM of tPA enzymatic activity. ***p < 0.001 versus OGD without NXY-059 by a Dunnet's post-hoc test after a significant ANOVA (p < 0.001). (b) Parallel measurement of the sucrose transport during the tPA transport experiment. Data are presented as mean ± SEM percentage of [14C]-sucrose transport. The 100% control value represents a Pe of 0.27 × 10− 3 cm/min obtained after 3 h normoxic conditions. ***p < 0.001 versus OGD without NXY-059 by a Dunnet's post-hoc test after a significant ANOVA (p < 0.001; n = 3 independent EC monolayers/ treatment). neuroprotection. However, since in vivo results from animal models of permanent ischemia and in vitro BBB results derived from OGD experiments also have clearly demonstrated that NXY-059 may access the CNS in pharmacologically significant concentrations, especially at the high free plasma concentrations used in vivo (Green et al., 2006), it has not been possible to rule out that NXY-059 may protect both brain endothelium and nervous tissue (neurons and glial cells). Recent results

4.1.

Materials

4.1.1.

Test compound and formulation

NXY-059 was supplied by AstraZeneca (AstraZeneca R&D Södertälje, Sweden). Due to the hygroscopic nature of the NXY-059 powder, its trihydrate form, NXY-643, was used for preparing solutions. The compound code NXY-059 is, however, used throughout the report. The compound was dissolved in HEPES-buffered Ringer solution (NaCl 150 mmol/L, KCl 5.2 mmol/L, CaCl2 2.2 mmol/L, MgCl2 0.2 mmol/L, NaHCO3 6 mmol/L, glucose 2.8 mmol/L, HEPES 5 mmol/L, H2O for injection). All reagents were obtained from Sigma.

4.1.2.

Reagents

All cell culture products, such as DMEM and FCS, were obtained from Invitrogen Life Technologies (Cergy Pontoise, France). Nylon filters BLUTEX T25 TOBLVM1 were obtained from SAATI (Sailly Saillesel, France).

4.2.

In vitro model of the blood-brain barrier

The method of Dehouck et al. (1990) was used with minor modifications. Bovine brain endothelial cells isolated from capillary fragments were cocultured with primary mixed glial cells from newborn Sprague–Dawley rats. The glial cells were isolated according to the method of Booher and Sensenbrenner (1972) and cultured for 3 weeks, plated on the bottom of cell culture clusters containing six wells each. The endothelial cells were seeded onto collagen-coated cell culture inserts, which were placed in the wells containing glial cells. The medium used for the coculture was Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 10%

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horse serum, 2 mmol/L glutamine, 50 mg/L gentamicin and 1 μg/L basic fibroblast growth factor (Sigma). The medium was changed every second day. Under these conditions, endothelial cells formed a confluent monolayer after 7 days. Experiments were performed 5 days after confluency.

4.3.

Oxygen glucose deprivation (OGD) studies

The medium of the coculture was changed every 2 days and experiments were performed 24 h after a medium change. On the day of the experiment, endothelial cells with or without glial cells were exposed to OGD conditions by adding a “serumlight” and low-glucose (0.2 g/L) medium equilibrated with nitrogen (OGD medium). Cells were then placed for 4 h in an oven at 37 °C with an atmosphere of 0% O2, 5% CO2, 95% N2 using Gas Pack Pouch bags (Becton Dickinson, Franklin Lakes, NJ, USA). Normal medium (brain capillary endothelial cell medium with 20% (v/v) serum) was used for coculture and for the experiments, the “serum-light” medium (5% (v/v) serum) was used to avoid temporary permeability increasing artifacts due to serum growth factors and to allow short studies. As controls, cells were placed in the “serum-light” medium (equilibrated with air, 1 g/L glucose) in normoxic conditions. In all experiments, the pH of the medium remained the same in normoxic or OGD conditions.

4.4.

Transport studies

4.4.1.

Transport studies in normoxia

Permeability studies were performed using either radiolabelled sucrose (Amersham, UK) or human recombinant tissue plasminogen activator (tPA) (Actilyse purchased from Boehringer Ingelheim, Paris, France). In these studies, endothelial cell monolayers were transferred to six-well plates containing 2.5 mL of Ringer–HEPES solution (150 mmol/L NaCl, 5.2 mmol/L KCl, 2.2 mmol/L CaCl2, 0.2 mmol/L MgCl2(6H2O), 6 mmol/L NaHCO3, 5 mmol/L HEPES, 2.8 mmol/L glucose, pH 7.4) per well, after the different treatments. 1.5 mL Ringer–HEPES solution containing [14C]-sucrose (Amersham, UK) and 20 mg/L tPA (only for tPA transport) was then added to the endothelial monolayer (upper compartment or luminal side). Incubations were performed at 37 °C. At 20, 40 and 60 min (for sucrose) and after 2 h (for tPA) aliquots were taken from each abluminal compartment; in addition, aliquots were taken from the stock solutions. The amount of radiotracer in the abluminal compartment was measured in a liquid scintillation analyser (Packard Instrument Company, Meriden, USA). The tPA activity in the abluminal compartment was determined using a fluorogenic substrate (Spectrozyme, XF444, American Diagnostica). For sucrose, triplicate control wells were also assayed to determine the endothelial permeability coefficient (Pe) calculated in centimeters per minute as described previously (Dehouck et al., 1992). In this calculation, both filter permeability (Psf = insert filter + collagen coating) and filter plus cell permeability (Pst= filter+ collagen + endothelial cells) were taken into account, according to the formula: 1 / Pe= 1 / Pst − 1 / Psf. For each experimental condition, data are mean values± SEM (n = 3 experiments) of radiolabelled sucrose Pe (in cm/min) and are expressed in % compared to control (a value of 100% is given to the control).

4.4.2.

151

Transport studies during OGD

OGD transport studies were performed as described above. For OGD studies, the Ringer–HEPES solution was first equilibrated with nitrogen and glucose was removed from the medium. Transport studies were performed in an airtight glove box filled with nitrogen at 37 °C (Forma Scientific, Mountain View, CA, USA).

4.5.

Fluorescent microscopy

4.5.1.

Treatment of endothelial cells with D-mannitol

To evaluate whether the fluorescent staining was sufficient to visualise a paracellular breakdown of the endothelial cells monolayer, mannitol, a mannose derived sugar alcohol commonly used to perform osmotic shock (Rapoport and Robinson, 1986), was used as a positive control for tight junction disruption. After 12 days of coculture, endothelial cells were separated from glial cells and subjected to 30 min mannitol exposure at a concentration of 1.4 mol/L before fixation.

4.5.2.

Occludin immunostaining

Filters with endothelial cells were fixed for 10 min in 1% (w/v) paraformaldehyde/PBS on ice and washed. Cells were permeabilized for 10 min with 0.1% (v/v) triton X-100/PBS, and pre-incubated for 30 min in 10% (v/v) normal goat serum/PBS. The specific primary rabbit polyclonal anti-human occludin (Zymed Laboratories Inc., San Francisco, CA, USA) was added (1/200 dilution in 2% (v/v) normal goat serum/PBS) for 60 min. After several washes with PBS, a secondary mouse anti-rabbit antibody (Alexa 568; Molecular Probes) was added for 60 min and filter sections were then mounted in Mowiol. Fluorescence was visualised with a Leica DMR fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and images were obtained with a Cool-Snap digital camera (Leica Microsystems).

4.6.

Statistical analysis

Three independent inserts were use for each experimental condition. The Dunnet's post-hoc test was used to assess differences between conditions after a significant ANOVA.

Acknowledgments This work was supported by grants from the Ministry of Research of France (fellowships to M. Culot and Ms Mysiorek). The authors thank Mrs A-M Lenfant for her technical assistance.

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