Blood–brain barrier permeability to the neuroprotectant oxyresveratrol

Neuroscience Letters 393 (2006) 113–118

Blood–brain barrier permeability to the neuroprotectant oxyresveratrol Christian Breuer, Gerald Wolf ∗ , Shaida A. Andrabi, Peter Lorenz, Thomas F.W. Horn Institute for Medical Neurobiology, Otto-von-Guericke University, Leipziger Strasse 44, Magdeburg D-39120, Germany Received 25 July 2005; received in revised form 8 September 2005; accepted 24 September 2005

Abstract We investigated to what extent the antioxidative hydroxystilbene oxyresveratrol (trans-2,3 ,4,5 -tetrahydroxystilbene, OXY), that we showed earlier to be strongly neuroprotective in a stroke model, may cross the blood–brain barrier (BBB) in healthy rats and in subjects submitted to focal infarction. Tissue extraction and in vivo microdialysis in the striatum show that systematically applied OXY is able to penetrate the BBB in control animals, but to a low extent. Microdialysis samples from animals that were subjected to a middle cerebral artery occlusion (MCAO) displayed strongly increased OXY levels (more than six-fold) in the infarct region as compared to sham-operated rats. Our data show that OXY may exert direct protective effects in the brain by crossing the BBB and may prove an excellent complementary drug for the treatment of neurodegenerative disorders that causally involve oxidative/nitrosative stress, especially in stroke. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Neuroprotection; Stroke; Hydroxystilbenes; Ischemia; Microdialysis; MCAO

Polyphenolic hydroxystilbenes are well known for their free radical scavenging properties [5,9]. One representative of this class, the naturally occurring OXY, despite its facile isolation from mulberry wood (Morus alba L.), is little investigated. Until now, data on OXY bioavailability and its brain concentrations upon in vivo applications, particularly information on its pharmacokinetics within an ischemic brain area, are incomplete. We reported earlier that OXY effectively scavenges hydrogen peroxide (H2 O2 ), nitric oxide (NO), and the artificial free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) [13]. Moreover, we observed that OXY selectively kills activated microglia which are assumed to aggravate the outcome of cerebral ischemia by a high NO-output pathway and the release of cytokines [11]. The high solubility of this drug in aqueous solutions and its otherwise low toxicity render OXY as a potential drug for the development of pharmalogical intervention in stroke therapy. However, when OXY was administered intravenously, only small amounts were detected in plasma [18]. Additionally, OXY is less lipophilic than other hydroxystilbenes like the much better studied Resveratrol [13,14]. Hence, the question arises whether sufficient amounts of OXY are crossing the BBB to explain the protective effects within the ischemic area. Since we

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0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.09.081

already showed previously that OXY application in an MCAO model of transient brain ischemia significantly reduced the brain infarct volume by approximately 54 and 63%, respectively [1], when compared to vehicle-treated MCAO rats, these effects may indeed be related to differences in BBB integrity or transport. To assess peripheral and brain tissue levels of OXY after its application in control and infarcted rats, we used two approaches: tissue extraction and in vivo microdialysis. Adult male Wistar rats, weighing approximately 290 g, were used. OXY (40 mg/kg body weight, prepared from mulberry wood as described previously [13]) and sodium fluorescein (FLUO, 62.5 mg/kg, Merck, Germany) were administered intraperitoneally (i.p.) dissolved in 20% 2-hydroxypropyl-␤cyclodextrine (Sigma–Aldrich, Germany) in a total volume of 1 ml/rat. One hour after injection the animals were anesthetized with Ketanest-S (Parke-Davis, Berlin, Germany)/Dormitor (Orinon Pharma, Finland) (3:1, v/v) and intracardially perfused with 0.9% saline. Reference blood samples were taken by cardiac puncture before starting the perfusion. Heparin, 75 IU (Thrombophob, Abbott, Germany) and 0.2 M phosphate buffer at pH 7.4 (NaPi) were added to the sample (2 ml NaPi/ml), and the mixture was centrifuged at 4000 rpm at 4 ◦ C for 5 min. The supernatants were aliquoted into vials and kept frozen till solid-phase extraction. After saline perfusion, the brain and liver samples were collected. NaPi was added to the samples (2 ml/g) and the mixture was centrifuged at 4000 rpm at 4 ◦ C for 15 min. OXY and

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FLUO were extracted from the supernatants by reversed phase extraction (Waters Oasis HLB cartridges, 1 cm3 , 30 mg). The cartridge was first conditioned with 1 ml of methanol, followed by 2 ml NaPi. One milliliter of each sample was loaded slowly onto the cartridge and rinsed with 2 ml of NaPi. The adsorbed fraction was eluted with 10 ml methanol and collected in a tube. The eluate solvents were evaporated under a nitrogen stream, redissolved in a mixture of acetonitrile/water (50:50, v/v) in a final volume of 1 ml and analyzed by HPLC. Brain sample volumes were reduced to 200 ␮l by vacuum centrifugation. Recovery was estimated by spiking control samples with known amounts of OXY and FLUO. The rats (290–330 g) were adapted to the laboratory conditions for at least 3 days, kept under a 12-h light/12-h dark cycle and given free access to food and water. Self-made ushaped microdialysis probes were implanted into the striatum 3 days prior to the experiment [8]. The tip of the microdialysis probe was placed in the striatum (coordinates were taken from [16]) within the coordinates 3 mm lateral to bregma and 5 mm intracerebrally. The animals were randomly divided into a sham and a group submitted to middle cerebral artery occlusion (MCAO). The probes were perfused with Ringer solution (Ringer-L¨osung Bernburg, Germany) and the flow rate was adjusted to 200 ␮l/h. Samples were collected in 30 min intervals. One hundred and twenty minutes after start of the sample collection, control animals (sham-operated group) were briefly anesthetized with 2% halothane in 50% N2 O/50% O2 . OXY (60 mg/kg body weight) and FLUO (0.185 mg/kg) were administered into the jugular vein in a total volume of 1 ml in 20% 2-hydroxypropyl-␤-cyclodextrine. Sample collection was continued for further 210 min after the injection. In another experiment for the purpose of assessing OXY levels in an ischemic brain infarct, animals were anesthetized with halothane and body temperature was controlled by a rectal probe throughout the whole surgical period and maintained at 37 ◦ C by a heating pad. Focal cerebral brain ischemia was induced by the intraluminal suture method [12] as described by [2]. Briefly, a 3-0 nylon suture (Ethicon, Brussels, Belgium) with its tip rounded by heating near a flame and coated with poly-l-lysine, was introduced into the internal carotid artery through a nick given in the external carotid artery and advanced 17–20 mm from the common carotid artery bifurcation to block the origin of middle cerebral artery (MCA). Immediately after occluding the MCA and wound suture, the animals were allowed to wake up and microdialysis perfusion was started to obtain control samples prior to the drug application. The intraluminal suture was left in place for 120 min while microdialysis samples were collected. After 120 min of occlusion, the intraluminal suture was gently removed under short halothane anesthesia to allow reperfusion. Simultaneously OXY and FLUO were administered into the jugular vein as described above and samples were collected for further 210 min. During the experiment the animals were closely observated and cerebral ischemia was verified by evaluating spontaneous motor activity (SPMA). Microdialysis samples were analyzed by HPLC within 3 days. The measured drug concentrations in the samples were

corrected by the recovery factor, which was determined by an in vitro microdialysis of different OXY and FLUO concentrations. Twenty-four hours after microdialysis the animals were kept under anesthesia (Ketanest-S/Dormitor 3:1, v/v) and intracardially perfused with 300 ml 4% paraformaldehyde. After decapitation the microdialysis probe was removed and the brain was extracted and post-fixed in paraformaldehyde overnight. The brain was then subbed in a 20% sucrose solution for 2 days and frozen at −80 ◦ C. The brains were cut on a cryostat into 20 ␮m coronal sections and collected in NaPi. At least 10 sections containing the lesion track of the microdialysis probe were carefully mounted onto polylysine-precoated slides and air-dried. A subset of the slides was then labeled with Nissl staining. The remaining slides were alternately labeled with immunofluorescent markers to illustrate the ischemic brain area. Therefore, DAPI (Boehringer, Germany) was combined with histochemistry either for OX-42 (purified mouse anti-rat monoclonal antibody 1:800, Pharmingen, San Diego), GFAP (rabbit polyclonal antibody 1:500, Progen, Heidelberg), or MAP2 (SMI 52 monoclonal mouse antibody 1:5000, Sternberger Monoclonals, MD, USA) according to standard staining procedure. Secondary antibodies were GFAP: Alexa Fluor 546 goat

Fig. 1. Levels of OXY and FLUO in blood plasma, liver, and brain of control animals 1 h after i.p. injection (40 mg/kg OXY, 62.5 mg/kg FLUO). Data represent mean + S.E.M. from four animals. OXY concentrations were 0.626 ± 0.059 ␮g/ml (plasma), 0.139 ± 0.007 ␮g/g (liver), and 0.038 ± 0.010 ␮g/g (brain). FLUO concentrations were 19.879 ± 1.915 ␮g/ml (plasma), 17.009 ± 0.652 ␮g/g (liver), and 0.399 ± 0.108 ␮g/g (brain).

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Fig. 2. Striatal in vivo microdialysis in sham-operated (n = 6) and MCAO (n = 5) rats. Samples were collected in 30 min intervals. The MCAO group was submitted to the vessel occlusion 120 min prior to the i.v. drug application (OXY 60 mg/kg; FLUO 0.185 mg/kg) in conjunction with reperfusion. Sham-operated rats received the same treatment except MCAO. After drug application samples were collected for further 210 min. Each point and vertical bar represent mean − S.E.M.

anti-rabbit IgG, MAP-2 and OX-42: Alexa Fluor 546 goat antimouse IgG (1:500, Molecular Probes, Eugene). After that half of the double-labeled sections were additionally stained with Fluoro-Jade C (FJ-C) as described by Schmued et al. [19]. The slides were then dried, cleared, and coverslipped with ImmuMount (Thermo Shandon, Pittsburgh). The fluorescent labels were visualized by standard fluorescent microscopy (Axiophot, Zeiss) and digitalized. OXY- and FLUO-concentrations in all collected samples were measured by HPLC, using an Waters Alliance 2695 separations module, 2487 Dual Absorbance detector, 474 Scanning Fluorescence detector and a Maisch Reprosil-Pur C18-AQ column (5 ␮m, 250 mm × 4 mm) with a 1 cm C18 precolumn. The injection volume was 80 ␮l, the temperature of the column oven 25 ◦ C. The mobile phase included solvent A (acetonitrile/water 10:90 (v/v), plus 82 mM citric acid, 25 mM ammonium acetate and 2 mM sodium chloride), solvent B (acetonitrile/water 50:50 (v/v), plus 82 mM citric acid, 25 mM ammonium acetate and 2 mM sodium chloride), solvent C (1 mM citric acid in methanol, titrated to pH 7.4 with 25% ammonium), and solvent D (10 mM citric acid in methanol:water (40:60, v/v), titrated to pH 7.4 with 25% ammonium). Separation of OXY and FLUO was effected with gradient elution (for details, see Table 1). UV-absorbance and fluorescence signals were recorded simultaneously for detecting OXY (λUV = 327 nm) and FLUO (λEX = 490 nm and λEM = 515 nm),

respectively. Standard injections indicated retention times of 14.3 min (OXY) and 34.0 min (FLUO). Limits of detection were 12 ␮g/l (OXY) and 40 ng/l (FLUO). Using the tissue extraction procedure in conjunction with the HPLC analysis protocol, blood plasma, liver and brain concentrations of OXY and FLUO were measured 1 h after i.p. injection of equal molar amounts (40 mg/kg OXY; 62.5 mg/kg FLUO). Recovery rates of 97 ± 1.8% (liver), 69 ± 3.9% (plasma), and 35 ± 6.9% (brain) for OXY and 75 ± 1.7% (liver), 71 ± 1.5% (plasma), and 29 ± 7.6% (brain) for FLUO were obtained and their concentrations were expressed as ␮g/ml (blood plasma) or ␮g/g tissue (liver or brain). As shown in Fig. 1, drug levels were highest in plasma. The lowest concentration of either drug was found in the brain. While the brain concentration of OXY reached approximately 6% of its plasma concentration, the corresponding brain tissue concentration of FLUO was only 2% of that in the plasma, despite far higher absolute plasma concentrations. A 2-h period of MCAO followed by reperfusion and i.v. application of OXY (60 mg/kg) and FLUO (0.185 mg/kg) resulted, in contrast to the sham operated group, in significant higher concentrations of both drugs in the striatal area of ischemia. Between 30 and 210 min after drug application, the average OXY concentration in sham-operated animals was 70.70 ± 11.49 nmol/l. In MCAO animals the average OXY concentration increased up to 439.65 ± 33.15 nmol/l in the dialysate (p < 0.01, Student’s

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Table 1 Parameters of the eluent gradient used for OXY and FLUO separation by HPLC Time (min)

Flow rate (ml/min)

Eluent A (%)

Eluent B (%)

Eluent C (%)

Eluent D (%)

00.00 19.50 20.00 22.00 24.00 46.00 49.00 51.00 55.00 57.00 60.00

1.00 1.00 0.70 0.70 0.70 0.70 0.70 0.70 0.70 1.00 1.00

90 40 40 0 0 0 0 90 90 90 90

10 60 60 0 0 0 0 10 10 10 10

0 0 0 0 0 100 100 0 0 0 0

0 0 0 100 100 0 0 0 0 0 0

t-test). The average FLUO concentration was 0.52 ± 0.07 nmol/l in the sham group and 1.88 ± 0.52 nmol/l in the MCAO group (p < 0.02). A large variability of the cerebral drug concentration course in the individual MCAO animals was observed (Fig. 2). A 6.6-fold (p < 0.01) increase in the concentration of OXY and a 3.9-fold (p < 0.04) increase of FLUO were found in animals subjected to ischemia/reperfusion injury compared to shamoperated control animals when concentrations were expressed as area under the curve (AUC, Fig. 3). Nissl staining was used for assessing the position of the microdialysis probe after the experiment. Size and location of the brain lesion were illustrated by sections that were immunostained for MAP-2 (neurons), GFAP (astroglia), and OX-42 (microglia). DAPI-staining was used as fluorescent nuclear stain, and general neuronal injury was visualized by FJ-Cstaining (Fig. 4). In the present study, we followed up on our earlier results on the neuroprotective properties of OXY [3,13] by assessing its permeability to brain tissue. The concentrations of OXY measured by tissue extraction 1 h after i.p. application in shamoperated animals were rather low in plasma and liver and showed even much lower levels in the brain. These findings correspond to former reports that show a low bioavailability of OXY and its metabolites after oral and intravenous application [18]. OXY’s high degree of hydroxylation points to a low BBB penetration rate if compared to other stilbenes [22]. Indeed, the observed brain OXY levels after tissue extraction were close to those measured for FLUO. FLUO was used as a hydrophilic control substance that binds to plasma proteins and crosses the BBB only to a minor extent [10]. As shown in the present study, it reaches far higher plasma levels compared to OXY. Others have also reported that upon systematical application, low amounts of FLUO are detectable in the brain either by brain microdialysis [21] or by tissue extraction methods [4]. Yet, one has to consider that low, but detectable FLUO concentrations within the brain tissue may be due to intraluminally trapped marker. In case of microdialysis, one has to take into account that traces of the substance may be taken up by the mechanical lesion of the tissue [7]. The low levels of OXY in brain tissue as seen in our non-ischemic animals suggest that OXY does not lend itself as a neuroprotective drug. However, our previous data describing the strong neuroprotective effects of OXY in MCAO rats [1] stand in

Fig. 3. Estimated concentration of OXY and FLUO in the striatum obtained by collecting microdialysis samples from 30 to 210 min after i.v. application of OXY (60 mg/kg) and FLUO (0.185 mg/kg), expressed as area under the curves (AUC) depicted in Fig. 2. OXYSHAM : 407.62 ± 59.98 nmol/l, 3.5 h; OXYMCAO : 2684.33 ± 150.41 nmol/l, 3.5 h; FLUOSHAM : 2.87 ± 0.23 nmol/l, 3.5 h; FLUOMCAO : 11.14 ± 3.63 nmol/l, 3.5 h; Sham group (n = 6); MCAO group (n = 5); Sham vs. MCAO, * p < 0.05; Sham vs. MCAO, ** p < 0.01 (data shown as mean + S.E.M., Student’s t-test).

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Fig. 4. (a) Scanned overview images of sections obtained from normal rats (normal) and sham-operated rats with microdialysis probe implantation (MPI). The third group (MPI + MCAO) was submitted additionally to MCAO. (b) First three rows of images depict the fluorescence signal of a nuclear staining (DAPI), evaluation of degeneration by Fluoro-Jade C (FJ-C) and the immunolabel for the microglial marker protein OX-42 (OX-42) for each treatment group (rectangle in (a)). The lower panel row shows the merged images of the three stainings (DAP + FJ-C + OX-42). Note that the lesion of the MPI + MCAO animal is located within an area that is intensively stained for OX-42 and with FJ-C but shows a reduced DAPI label compared to MPI animals due to the infarcted tissue. Data of GFAP and MAP-2 staining is not shown.

strong contrast to this assumption. The question arose, hence, if OXY may display different pharmacokinetics in post-ischemic brain tissue. To answer this question more precisely we used in vivo microdialysis to monitor the extracellular concentrations of the applied drugs. Indeed, using this technique in conjunction with the MCAO procedure, we showed that the brain OXY levels found in ischemic tissue of MCAO rats were increased to approx. 660% in comparison to sham-operated animals when OXY was applied i.v. at the time of reperfusion. This finding indicates that OXY readily enters the extracellular fluid of the brain

parenchyma when the drug is applied at the time of reperfusion. The simultaneously applied FLUO that served as an indicator for the BBB integrity [15], showed as well an increased concentration by approx. 390%, but to a lesser extent in the dialysates after reperfusion, indicating a breakdown of the BBB due to the MCAO-induced ischemic insult [17]. The enrichment of drugs in ischemically affected brain areas may find an additional explanation by a malfunction of multidrug-resistant associated transport proteins [20]. The MCAO model is a highly suitable paradigm to assess pharmacological properties of drugs because it resembles

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various pathophysiological characteristics of stroke in human subjects including thrombolysis as mimicked by reperfusion [6]. Hence, the time point of reperfusion, at which we chose to apply OXY, is one of clinical relevancy since it constitutes the commencement of post-insult therapy and may be, therefore, also the first realistic chance for additional neuroprotective peri-thrombolytic care. Moreover, the MCA, the specific occlusion site in this model, is the most commonly affected vessel in human stroke [12]. As shown above, tissue extraction revealed that in the nonischemic brain the brain/plasma concentration ratio is higher for OXY than for FLUO. This implies that OXY may better cross the BBB than the control substance FLUO, a difference that is much more pronounced in the microdialysis experiments. In particular, after the ischemic insult was the OXY/FLUO ratio in the microdialysis samples strongly increased indicating a facilitated BBB penetration of OXY. Taken together, we adapted a tissue extraction protocol and the in vivo microdialysis technique in conjunction with HPLC analysis for the determination of extracellular drug levels in ischemic and non-ischemic animals to assess the permeability of the BBB to the neuroprotectant OXY. We present evidence that, although little OXY was found in the brain under physiological conditions, its levels increase dramatically after ischemia/reperfusion injury. Such improved drug availability allows this drug to directly exert its neuroprotective effects within the ischemic tissue after the insult. Therefore, we suggest that OXY may prove an excellent complementary drug for the treatment of neurodegenerative disorders that causally involve oxidative/nitrosative stress, especially in stroke. One future goal may be to widen the therapeutic window by enhancement of drug transport across the BBB. Acknowledgments FJ-C was a generous gift of L. Schmued [19]. The study was financially supported by the grant of the Kultus-Ministerium of the Land Sachsen-Anhalt (3T213/0703M). References [1] S.A. Andrabi, M.G. Spina, P. Lorenz, U. Ebmeyer, G. Wolf, T.F. Horn, Oxyresveratrol (trans-2,3 ,4,5 -tetrahydroxystilbene) is neuroprotective and inhibits the apoptotic cell death in transient cerebral ischemia, Brain Res. 1017 (2004) 98–107. [2] L. Belayev, O.F. Alonso, R. Busto, W. Zhao, M.D. Ginsberg, Middle cerebral artery occlusion in the rat by intraluminal suture. Neurological and pathological evaluation of an improved model, Stroke 27 (1996) 1616–1622 (discussion 1623). [3] P.H. Chan, Reactive oxygen radicals in signaling and damage in the ischemic brain, J. Cereb. Blood Flow Metab. 21 (2001) 2–14.

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