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Research Report

Quantitative evaluation of blood–cerebrospinal fluid barrierpermeability in the rat with experimental meningitis usingmagnetic resonance imaging

Hiroyuki Ichikawaa,⁎, Makoto Ishikawaa, Mari Fukunagaa,Koichi Ishikawab, Hironobu Ishiyamaa

aThird Institute of New Drug Discovery, Otsuka Pharmaceutical Co., Ltd., Tokushima, 771-0192, JapanbInstitute for Molecular and Cellular Regulation, Gunma University, Maebashi, 371-8512, Japan

A R T I C L E I N F O

⁎ Corresponding author. Third Institute of NTokushima, 771-0192, Japan. Fax: +81 88 665

E-mail address: hichikawa@research.otsuAbbreviations: MRI, magnetic resonance

gadolinium–diethylene triamine pentaacetic

0006-8993/$ – see front matter. Crown Copyrdoi:10.1016/j.brainres.2010.01.050

A B S T R A C T

Article history:Accepted 16 January 2010Available online 28 January 2010

Disruption of the blood–brain barrier (BBB) and/or the blood–cerebrospinal fluid barrier(BCSFB) is thought to be one of the major pathophysiological consequences of meningitisand contributes to the development of adverse neurological outcomes. In order to clarifythis hypothesis further, we sequentially quantified the permeability of these barriers withmagnetic resonance imaging (MRI) contrast enhancement using gadolinium–diethylenetriamine pentaacetic acid (Gd–DTPA) in rats with various experimentally-inducedmeningitis. Meningeal inflammation was elicited by an intracisternal injection ofinterleukin (IL)-1β, prostaglandin (PG) E2, or lipopolysaccharide (LPS). Barrier permeabilitywas calculated from the gadolinium-enhancement ratio (GER) in the subarachnoid space(SAS). The secretion of Gd–DTPA into the SAS was monitored by T1-weighted imaging afteran intravenous injection of Gd–DTPA. As a significant linear correlation was observedbetween the GER and the standard solution, the concentration of the secreted Gd–DTPAwere determined from the GER. The maximal intensity in SAS was detected at 5 min afterGd–DTPA administration and it declined gradually. Among the inflammatory agents, IL-1βwas found to induce the most severe meningitis as determined from the GER. Theconcentration of Gd–DTPA in the SAS increased in a dose-dependent manner followingIL-1β intracisternal injection. On the other hand, no significant changes in signal intensityof the brain parenchymal areas due to IL-1β injection were observed. The findings suggestthat the permeability of the BCSFB can be evaluated quantitatively by calculating the GER.MRI with Gd–DTPA provides a useful method to monitor the change in the permeability tothe brain barriers.

Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.

Keywords:Blood–cerebrospinal fluid barrierPermeabilityMagnetic resonance imaging

ew Drug Discovery, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno, Kawauchi-cho,6976.ka.co.jp (H. Ichikawa).imaging; BBB, blood–brain barrier; BCSFB, blood–cerebrospinal fluid barrier; Gd–DTPA,acid; GER, gadolinium-enhancement ratio; SAS, subarachnoid space

ight © 2010 Published by Elsevier B.V. All rights reserved.

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1. Introduction

The blood–brain barrier (BBB) and the blood–cerebrospinal fluidbarrier (BCSFB) are composed of endothelial cells and glial cells,which restrict the traffic of molecules in and out of the centralnervous system (CNS). As such, each barrier provides homeo-stasis and protection to the brain and the spinal cord (Mayhan,2001; Weiss et al., 2008). Changes in the permeability of the BBBand/or the BCSFB are supposed to contribute to the pathology ofcertain neurological diseases with a known inflammatorycomponent, suchasmeningitis, stroke, andAlzheimer's disease(Weiss et al., 2008; Boje, 1995; Guoet al., 2008). Inflammation cancause disruption of the barriers, further compromising theintegrityof theCNS.Analysisof thebarrierpermeability isoneofthe major issues for the understanding of many neurologicalevents. Clarifying themechanisms bywhich the function of thebrainbarrier is altered in these diseases appears to be importantfor thedevelopment ofnew treatment strategies and to improveboth their short- and long-term prognoses. So far, Evan's bluedye, horseradish peroxidase (HRP) and radioactive tracers havebeen used to evaluate the permeability of the barrier (Uyamaet al., 1988; Tengver, 1986; Dobbin et al., 1989). However, noneof these tracers can be visualized by conventional imagingtechniques. That is, the experimental animals have to besacrificed to observe the distribution of the tracers. Therefore,chronological observation of the barrier disruption in the sameanimal is impossibleby conventional techniques. Especially, thechanges in the permeability of the BCSFB during inflammationhave not been adequately studied.

Magnetic resonance imaging (MRI) with gadolinium–diethy-lene triamine pentaacetic acid (Gd–DTPA) enhancement hasbeen widely used for detecting the various pathological lesions

Fig. 1 – GER images obtained from the brain of control rat after Gbrain obtained 5, 15, and 30 min after intravenous Gd–DTPA admSAS (B).

and abnormal vasculature in the CNS, such as stroke andinfections. Gd–DTPA is a paramagnetic contrast agent that hasbeen shown to enhance T1-weighted (T1W) MRI of variouslesions of the central nervous system (Maravilla, 2006). Intrave-nously-injected Gd–DTPA accumulates in regions of disruptionand causes a local increase in the MRI signal intensity seen onT1W images. Gd–DTPA cannot cross the ventricle–brain barrieror the blood–ventricle barrier because of its charge and highmolecular weight (Bronen and Sze, 1990). Thus the dilution rateof Gd–DTPA by freshly secreted cerebrospinal fluid (CSF) couldbe obtained from the decay of T1W images of the CSF. In MRI,so far, it is possible to detect the amount of leakage of Gd–DTPAby the signal intensity. However, it is difficult to measure theconcentration of Gd–DTPA quantitatively.

In the present study, we have used MRI with Gd–DTPAenhancement in order to sequentially quantify the perme-ability of the barriers. The Gd–DTPA concentrations of theareas alongwith the BCSFB and the parenchymal areas (whichare under the BBB) were determined before and after the injec-tionof LPS, IL-1β, or PGE2 into the rat brain, by analyzing thepreand post T1W images.

2. Results

2.1. Serial MRI images of experimental meningitis

T1W and GER images obtained from the brain of the control ratbefore and after Gd–DTPA administration are shown in Fig. 1.The slight increase in signal intensity of GER images wasobserved in the SAS and the cerebral ventricle, but not in thebrain parenchymal areas (Fig. 1A). The maximal signalintensity was observed at 5 min, and it gradually decreased

d–DTPA administration. T1W and GER images of a normal ratinistration (A). The region of interest (ROI) was put in the

Fig. 2 – GER images in the IL-1β-treated rat and the controlrat. GER images shows the increase in the signal intensity(5 min after Gd–DTPA administration) from the SAS and thecerebral ventricular space after administration of IL-1β(50,000 U/mL) into the intracisternal cavity.

Fig. 3 – Serial GER images of the brain taken after IL-1β, LPS, andIL-1β (50,000 U), LPS (200 µg), or PGE2 (10 µg) was administered intreatment, Gd–DTPA (0.2 mmol/kg) was administered via the introbtained from the same brain.

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afterwards in both the SAS and the cerebral ventricle. Therewere no significant changes in other brain areas. Typical GERimages from the IL-1β-treated and the control rats (5 min afterGd–DTPA administration) are shown in Fig. 2. The signalintensities from the SAS and the cerebral ventricular space ofthe IL-1β-treated rats were significantly greater than those ofthe control rats. On the other hand, no significant changesin the signal intensity of the brain parenchymal areas due toIL-1β injection were observed (Fig. 2).

2.2. Evaluation of BCSFB permeability using GER duringexperimentally-induced meningitis

The ROI was put in the SAS to evaluate the permeability of theBCSFB (Fig. 1B). Themaximal intensity in the SASwas detectedat 5 min after Gd–DTPA administration and then it graduallydeclined (Fig. 3) in all groups with meningitis. The GER in

PGE2 inducedmeningitis following Gd–DTPA administration.to the intracisternal cavity of rat. Four hours after eachavenous vein. After 5, 15 and 30 min, GER images were

Fig. 5 – WBC infiltration into CSF following intracisternalinjection of IL-1β. IL-1β (25,000 and 50,000 U) wasadministered into the intracisternal cavity of rats. Four hoursafter the treatment, CSF was aspirated from the cisternamagna. CSF was used to count WBCs by the trypan blueexclusion. (n=5). The results represent mean±S.E. WBCs inCSF were not detected in the control rats.

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the SAS almost returned to the baseline values 30 min afterGd–DTPA administration. The concentrations of Gd–DTPA inthe SAS of the 25,000 U/mL-IL-1β-treated and the 50,000 U/mL-IL-1β treated groups were 96.2±4.5 µM and 122.4±5.9 µM,respectively 5 min. The Gd–DTPA concentration of the controlgroup was 36.4±4.2 µM at 5 min (Fig. 4). The rise in Gd–DTPAconcentration seemed to be dependent on the dose of IL-1β.The Gd–DTPA concentrations from the LPS-treated and thePGE2-treated groups were 90.1±10.4 µM and 53.8±3.6 µM,respectively 5 min after Gd–DTPA administration (Fig. 4).Among the inflammatory agent, IL-1β was found to inducethe most severe meningitis as judged from the GER. IL-1βcaused the increase in the signal intensity also in the cerebralventricle. On the other hand, the changes in the signal inten-sity in the brain parenchyma were very little, as compared tothose in the SAS and cerebral ventricle.

2.3. Infiltration of WBCs into the CSF

Pleocytosis was observed in the CSF at 4 h after intracisternalinjection of IL-1β. The infiltration of WBCs into the CSF of the25,000 U/mL-IL-1β-treated and the 50,000 U/mL-IL-1β-treatedgroups were 3.15±0.50×103/mL and 4.05±0.74×103/mL,respectively (Fig. 5). WBCs were not detected in the CSF of thecontrol group.

2.4. Protective effect of prednisolone on theBCSFB disruption

The protective effect of prednisolone on the BCSFB disruptioninduced by IL-1β is shown in Fig. 6. Prednisolone significantlyinhibited the Gd–DTPA leakage into the SAS induced by IL-1β

Fig. 6 – Protective effect of prednisolone on the BCSFBdisruption in the IL-1β-induced meningitis. Prednisolone(3 mg/kg) was administered orally to the rat 30 min before theintracisternal injection of artificial CSF containing IL-1β(25,000 and 50,000 U/site). Four hours after the IL-1βtreatment, Gd–DTPA (0.2 mmol/kg) was administered via theintravenous vein. T1W images were measured after 5, 15,and 30 min (n=5). Gd–DTPA concentration was calculatedfrom the GER. The differences between the prednisolonetreated and the control group were assessed by one-wayANOVA followed by the two-tailed Dunnett's test. Theresults represent mean±S.E. *P<0.05 versus each controlgroup.

Fig. 4 – Gd–DTPA concentration in the subarachnoid spacedetermined by GER of IL-1β, LPS, and PGE2 induced ratmeningitis. IL-1β (25,000, 50,000 U), LPS (200 µg), or PGE2(10 µg) was administered into the intracisternal cavity of rat.Four hours after each treatment, Gd–DTPA (0.2 mmol/kg) wasadministered via the intravenous vein (n=5). After 5, 15, and30 min, Gd–DTPA concentrationwas calculated from the GER.The differences between the treated and the control groupwere assessed by one-way ANOVA followed by thetwo-tailed Dunnett's test. The results represent mean±S.E.*P<0.05 versus each control group.

Fig. 7 – Correlation between gadolinium–diethylenetriamine pentaacetic acid concentration and GER. Gd–DTPAworking standard solutions were prepared from artificialcerebrospinal fluid (final conc. 0, 50, 75, 100, 150, and200 µg/mL). The MR images were obtained using a 62 mminner diameter quadrature coil (Varian, Inc., Palo Alto, CA.USA). T1W images were obtained using the same parametersas for the brain. GER was calculated from the formula:GER = [(T1WMRIconc. 50−200−T1WMRIconc. 0) / T1Wconc. 0] × 100.

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(Vehicle 137.7±7.4 versus IL-1β+Prednisolone 70.2±7.2) at5 min.

2.5. Correlation between GER and Gd–DTPA concentration

Fig. 7 shows the correlation between the GER and the Gd–DTPAconcentration. A significant linear correlation was observedbetween the GER and the Gd–DTPA concentration (correlationcoefficient: y=0.89×−0.71, R2=0.996). This indicates that theGER value can be used to quantitatively evaluate the perme-ability of the BCSFB.

3. Discussion

A novel aspect of this study was the application of GER for thequantitative analysis of BCSFB leakage of the Gd–DTPA. Thisapproach revealed a linear correlation between the GER andthe Gd–DTPA concentration (Fig. 7). Therefore the applicationof GER showed the differences in the permeability of theBCSFB by IL-1β dose-dependently and by the inflammatoryagents such as IL-1β, PGE2 and LPS. Moreover, GER showed theprotective effect of prednisolone on the BCSFB disruptioninduced by IL-1β. The permeability of the BCSFB in previousstudies has been evaluated by the signal intensity of Gd–DTPAenhanced T1W images or by the T1 value of CSF after adminis-tration of Gd–DTPA (Nagahiro et al., 1994; Meltzer et al., 1996;Thwaites et al., 2007). In addition, Gd–DTPA has been reported

to be useful to visualize the changes in the BBB permeability inmeningitis and the lung permeability in pneumonia (Speller-berg et al., 1995; Idänpään-Heikkilä et al., 1997). However thesignal intensity of T1W images does not afford the quantitativeanalysis of BCSFB leakage of the Gd–DTPA, although it affordsthe relative difference of the signal intensity. On the contraryT1 value can afford the quantitative analysis of BCSFB leakageof the Gd–DTPA because T1 value depends on the gadolinium–diethylene concentration. However the accurate measure-ments of T1 value require several tens of minutes.

In the present study, to evaluate the permeability of theBCSFB, we put the ROI in the SAS. We chose this structure as itis rather stable, in terms of morphology, during pathologicalconditions (Fig. 1B), although the volume of this space is verysmall. The error due to the partial-volume effect in this spaceis smaller than that in the cerebral ventricle. The spatialresolution of the T1W images was 0.049 mm for the evaluationof BCSFB permeability in the SAS space. Thus, we could eval-uate the permeability of the BCSFB in the SAS using the highspatial resolution.

In this study, experimental meningitis was induced byinflammatory agents such as IL-1β, PGE2, and LPS. Our analysiswith the GER images showed that there were differences ofBCSFB permeability induced by IL-1β, PGE2, or LPS (Fig. 4). Thepresentdata clearly showed that the leakageofGd–DTPAoccursin the SAS and its surrounding ventricle area duringmeningitis.

After the intravenous administration of Gd–DTPA, bloodconcentration of Gd–DTPA fell progressively decreasing. Similarpatterns have been reported in many tissues, including thespleen, the liver and thebrain (Deanetal., 1988).Our resultsmayindicate that the clearance patterns of Gd–DTPA in the SAS aresimilar to those in such tissues.

Themechanismunderlying this can be explained as follows:Gd–DTPAmaymigrate into the ventricle via the choroid plexusdue to the increased permeability of the BCSFB, when menin-gitis occurs. Parenchymal leakage was not seen in any experi-mental rats until at least 30min after Gd–DTPA injection.Therefore, it may be suggested that the BCSFB, rather than theBBB, plays a key role in the influx of blood factors into the brainduring meningitis.

In the present study the increase in the BBB permeabilityinduced by inflammatory agents was not observed. This maybe due to the evaluation period after administration of inflam-matory agents. The parenchymal damage in experimentalmeningitis induced by bacteria (including LPS) or inflamma-tory agents have been reported in many studies. In suchstudies, the assessment of the BBB integrity has been carriedout 8–40 h after bacteria injection (Leib et al., 2000; Sellner andLeib, 2006; Meli et al., 2006; Grandgirard et al., 2007; Jaworowiczet al., 1998). Moreover, in the IL-1β-induced meningitis,substantial changes in the BBB permeability assessed by MRIwere not observed at 3 h, but clear enhancementwas observedat 5–6 h after the IL-1β injection into the brain (Blamire et al.,2000). In the present experiment, we have evaluated the BBBpermeability until 4 h after the IL-1β injection. Thus, thedetection of BBB disruption might be possible at later than 4 hafter the injection. Another explanation may be due to theage-related effects of IL-1β. The IL-1β injected into the brain ofjuvenile rats produced the disruption not only of the BCSFBbut also of the BBB at 4 h after IL-1β injection. On the contrary

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in the adult rats the breakdown did not occur in the BBB, but inthe BCSFB (Anthony et al., 1997). We have used adult rats inthis experiment. This observation appears to accord with ourresult.

During meningitis, bacterial endotoxins such as LPS causethe induction and expression of various cytokines (Smithet al., 1993), which initiate a host defense response viaelaboration of various inflammatory mediators. Such factorscontribute to meningeal inflammation and the subsequentdisruption of the BBB and the BCSFB (Tunkel and Scheld, 1993).IL-1β has been reported to be intimately involved in thedisruption of the brain barrier (Bronen and Sze, 1990; Tunkeland Scheld, 1993). Similarly, PGE2 is thought to play an impor-tant role in the development of meningeal inflammation andmediates vasodilation, increased vascular permeability, andedema (Milstien et al., 1994; Roos, 1995). Among the inflam-matory agents used in this study, IL-1βwas themost effective.This may indicate that LPS is involved in IL-1β secretion at theBCSFB. PGE2 dosed intracisternally elicited disruption of theBCSFB (Kadurugamuwa et al., 1989). The disruption of theBCSFB by PGE2 seems to be very weak.

It has been previously reported that glucocorticoid canprevent the alteration of tight junction and the barrier dys-function induced by Streptococcus (Tenenbaum et al., 2009). Weconfirmed the protective effect of prednisolone (a gluco-corticoid) on the increase in the BCSFB permeability inducedby IL-1β. Prednisolone has been found to inhibit significantlythe IL-1β action on the BCSFB (Fig. 5). These findings maysuggest that the inhibitory action of steroid may be involvedin its clinical potential in the treatment of meningitis.

Evan's blue dye, HRP, and radioactive tracers have beenexploited in various permeability assays. Evan's blue dye hasbeen bound to bovine albumin in order to measure thepermeability of the brain barriers (Uyama et al., 1988). BecauseEvan's blue dye has been used in a colorimetric assay, there isthe inherent possibility of interference due to the contamina-tion of the CSF with the blood or serum samples. Fluorescei-nated dextrans, horseradish peroxidase, and Evan's blue dyehave a common disadvantage of being poorly quantitative.The present findings may suggest that MRI is a useful tool forquantifying BCSFB permeability with several advantages overother assays.

In conclusion, the quantification of the changes in thepermeability of theBCSFBwasmeasured in ratswithmeningitisusing Gd–DTPA enhanced T1W images. GER measured withMRI appears to be a useful and non-invasive technique for therepeated in vivo measurements of various physiological andpathological conditions.MRI is, therefore, of significantvalue forthe evaluation of successful therapeutic approaches.

4. Experimental procedures

4.1. Materials

Prostaglandin E2 (PGE2) and lipopolysaccharides (LPS; E. coliserotype 055:B5) were purchased from Sigma Chemical Co.(St. Louis, MO, USA). Human recombinant IL-1β (2×107 U/mgprotein) was synthesized at the Otsuka Pharmaceutical Co.,Ltd. (Tokushima, Japan). Gd–DTPA dimeglumine (Magnevist®)

was purchased from Bayer Schering Pharma (German). Pred-nisolone was purchased fromWako Pure Chemical industries,Ltd. (Japan).

4.2. Experimental animals

Fifty adult rats were used for the present study. The experi-ments were conducted under the Guidelines for AnimalCare and Use of Otsuka Pharmaceutical Co., Ltd. Male Wistarrats (8–10 weeks old) were purchased from Charles River Japan.Ratswerehoused ina room(21–24 °C) thatwas lit for 12 h (07:00–19:00) daily. Rats were allowed free access to Otsuka's in-housetap water and pellet food (MF, Oriental Yeast, Tokyo, JAPAN).

4.3. Induction of experimental meningitis

Twenty-five rats were used for the meningitis experiment. Onthe experimental day, the rats were anesthetized with Avertin(1 mL/100 g) and placed in a stereotaxic frame. Using a 25 µLHamilton microsyringe, 5 µL of artificial CSF containing IL-1β(25,000 and 50,000 U), LPS (200 µg), or PGE2 (10 µg) wasadministrated to the intracisternal cavity (n=5), as previouslydescribed (Anthony et al., 1997; Boje et al., 2003). The controlrats were injectedwith the same volume of artificial CSF (n=5).The artificial CSF was composed of 120 mM NaCl, 3.0 mM KCl,1.2 mM CaCl2, 1.2 mM MgCl2, 0.67 mM NaH2PO4, and 0.3 mMNa2HPO4, pH7.4. Four hours after each treatment, Gd–DTPA ata dose of 0.2 mmol/kg was administrated via the intravenousvein.

4.4. Influence of IL-1β on CSF exudation of whiteblood cells

Fifteen rats were used to investigate the WBC infiltration intoCSF. CSF samplewas obtained 4 h after intracisternal injectionof IL-1β (25,000 and 50,000 U/site), (n=5). The control ratswere injected with the same volume of artificial CSF. The CSFwas collected by the aspiration from the cisterna magna viapuncture of the allanto-occipital membrane. The CSF sampleswere used to count WBCs by the trypan blue exclusion.

4.5. Protective effect of prednisolone on the BCSFBdisruption in IL-1β induced meningitis

Ten rats were used to evaluate the protective effect ofprednisolone on the IL-1β-induced BCSFB disruption. Prednis-olone was suspended in 0.5% carboxymethyl cellulose (CMC)at a concentration of 0.6 mg/mL and was administered orallyto the rat 30 min before the intracisternal injection of artificialCSF containing IL-1β (50,000 U/site), (n=5). CMC (0.5%) solutionwas administered for the control group (n=5).

4.6. MRI measurements

Rats were anesthetized by inhalation of 1–1.5% halothane in aN2/O2 (70:30) mixture through a facemask and were placed in astereotaxic head holder during the MRI measurements. MRImeasurements were performed using the Inova 300 ImagingSystem (7 T) with the VNMRj 1.1d software (Varian, Inc., PaloAlto, CA.USA). A volumecoil and surface coil (RAPIDBiomedical

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GmbH, Germany) were used for signal transmission and detec-tion, respectively. (T1W images repetition time (TR), 500 ms;echo time (Te), 10 ms; number of scans, 2; slice thickness, 2 mm;matrix size, 256×256; the images were zero-filled to 512×512;and the field of viewwas 25×25mm) were obtained before, andat 5, 15, and 30min after the administration of Gd–DTPA. Theregion of interest (ROI) was put in the subarachnoid space.Gadolinium-enhancement ratio (GER) images were calculatedfrom the formula:

GER = T1W MRI post contrast – T1W MRI pre contrastð Þ½= T1W pre contrast� × 100:

Thus, the pixel-intensities displayed the % signal increasedue to Gd–DTPA leakage.

4.7. Correlation between GER and Gd–DTPA concentration

The samples of Gd–DTPAwere sequentially diluted in artificialCSF (final conc. 0, 50, 75, 100, 150, and 200 µg/mL) in order toobserve the correlation between the GER and the Gd–DTPAconcentration. The MR images were obtained using a 62 mminner diameter quadrature coil (Varian, Inc., Palo Alto, CA.USA). T1W imageswere obtainedusing the sameparameters asfor the brain. GER was calculated from the formula:

GER = T1W MRIconc: 50−200– T1W MRIconc:0ð Þ= T1Wconc: 0� �

× 100:

4.8. Statistical analysis

Results were expressed as mean±S.E. Statistical analysis wasperformed with SAS (SAS Institute Japan Ltd., R8.1). The differ-ences between the treated and control groups were assessed byone-wayANOVA followedby the two-tailedDunnett's test,withstatistical significance at P<0.05.

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