Ž .Brain Research 761 1997 4–10

Research report

Evidence for pore-like opening of the blood-brain barrier following forebrainischemia in rats

Edward Preston a,), David O. Foster b

a Institute for Biological Sciences, National Research Council of Canada, Building M54, Ottawa, Ont., K1A 0R6, Canadab Institute for Biodiagnostics, National Research Council of Canada, 435 Ellice AÕe., Winnipeg, MB, R3B 1Y6, Canada

Accepted 4 March 1997

Abstract

Ž .The nature of the delayed blood-brain barrier BBB opening that occurs in rats subjected to forebrain ischemia by the technique ofŽ . Ž .two-vessel carotid occlusion plus hypovolemic hypotension 2VO ischemia was probed by examining the simultaneous, trans-barrier

Ž .movement of two hydrophilic, normally poorly permeative solutes of markedly different molecular size: sucrose MWs342 and inulinŽ . Ž . ŽMWf5000 . Pentobarbital-anesthetized, male, Sprague–Dawley rats 342–374 g were subjected to 10 min of 2VO ischemia tympanic

. w14 xtemperature, 37.5–38.08C ; 6 h later they were reanesthetized and, along with non-ischemic controls, injected i.v. with C sucrose andw3 x Ž . Ž .H inulin. Transfer constants K s for blood-to-brain movement of the tracers and V s apparent initial volumes of tracer distributioni i

Ž .were determined for six brain regions by the multiple-time, graphical method tracer circulation times from 3 to 30 min . V s differed littlei

or insignificantly between the two tracers, or between control and post-ischemic rats; the values did not suggest appreciable endothelialŽ y1 y1.binding of either tracer that might lead to its uptake by adsorptive-phase endocytosis. In the controls, regional K s"S.E.M. nl g si

Ž . Ž .for inulin ranged from 0.18"0.04 to 0.31"0.09 and were significantly lower P-0.01 than K s for sucrose 1.53"0.16–1.91"0.29 .iŽ . Ž .The K ratio sucroserinulin across brain regions mean, 6.6; S.E.M., 0.6 was much lower than would be expected according to thei

concept that movement of most organic non-electrolytes across the intact BBB occurs by dissolution in and diffusion through endothelialcell plasma membranes, at a rate proportional to the lipid solubility and diffusivity of the solute. This finding is interpreted as indicating

Ž .that a portion of the transfer of sucrose and inulin occurred by a mechanism other than dissolution-diffusion e.g., via pores or vesicles .Ž .In the post-ischemic rats, K s for both tracers were elevated significantly P-0.01 in parietal cortex, striatum, hippocampus, andi

Ž . Ž y1 y1.midbrain. The post-ischemic increases DK s in these regions were greater for sucrose 1.90–3.31 nl g s than for inuliniŽ . Ž .0.80–1.33 . Across brain regions the ratio between sucrose DK and inulin DK averaged 2.9 S.E.M., 0.2 , a value significantly greateri i

than the ratio of 1 that would be expected were the BBB opening due to an enhancement of micropinocytosis and vesicular transport. TheŽ .correspondence of the mean DK ratio with the ratio of the free diffusion coefficients of the tracers D rD s2.9; water, 388Ci f, suc f, inu

suggests that the delayed opening of the BBB following 2VO ischemia involves the formation of trans- or paracellular, aqueous pores orchannels.

Keywords: Blood-brain barrier; Cerebral ischemia; Stroke; Transfer constants; Permeability-area product; Sucrose; Inulin

1. Introduction

Ž .In the 2VO two-vessel occlusion rat model of globalw xbrain ischemia 20 a combination of temporary, bilateral,

carotid artery occlusion and temporary blood withdrawalŽ .to produce arterial hypotension -50 mmHg effects deep,

forebrain ischemia, which results in regionally selective,delayed neuronal death in the striatum, hippocampus and

w xneocortex 20 and associated injury to the blood-brain

) Ž .Corresponding author. Fax: q1 613 941-4475; e-mail:ed.preston@nrc.ca

Ž . w xbarrier BBB 14,15 . The time course of BBB openingafter 2VO ischemia has been examined in our laboratory

w3 xusing H sucrose and the two-compartment model forw xradiotracer assessment of BBB permeability 14 . It was

found that 10 min of 2VO ischemia results in increases ofŽ .severalfold in the transfer constant K for blood-to-braini

permeation of sucrose in striatum and hippocampus, andsmaller increases in K in other brain regions. The open-i

ing of the BBB was pronounced at 6 h after the ischemiaand was largely reversed within 24 h, although neuronaldeath in selectively vulnerable regions does not become

w xevident until 1–3 days post ischemia 6,16,20 . Similar

0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 97 00323-5

( )E. Preston, D.O. FosterrBrain Research 761 1997 4–10 5

temporal changes in cortical BBB permeability followingw xglobal brain ischemia were reported by Dobbin et al. 2

w14 xwho used a 4VO rat model and C aminoisobutyric acidŽ .AIB as the tracer. The nature of the ischemic injury tothe BBB and its relationship to the neurochemical distur-bances preceding the delayed neuronal death have not yetbeen determined.

The present study explored the physical nature of theischemic opening of the BBB by examining alterations inBBB permeability to two normally poorly permeative so-

w14 x Žlutes of markedly different size: C sucrose MWs342,˚ 3. w x Žradiuss4.6 A and H inulin MWf5000, radiusf13

˚ .A . An analogous approach employing these two soluteshad been used previously for examining the nature of

w xosmotic opening of the BBB 24 , the idea being todistinguish between formation of pores and stimulation ofvesicular transport as the mechanism of increased BBBpermeability. BBB opening due to stimulation of pinocy-totic activity and vesicular transport in endothelial cellsshould affect the permeation of both tracers to a similarextent, since both have molecular dimensions much smallerthan endothelial vesicles, which have radii in the order of

˚ w x250 A or more 19,22 . If, on the other hand, BBB openingwere due to formation of aqueous pores or channels within

Žor between endothelial cells e.g., by widening of the.normally tight junctions between endothelial cells differ-

ential effects on the permeation of sucrose and inulinwould be expected as a consequence of the lower diffusiv-ity of the larger molecule and likely also from greatersteric hindrance of its diffusion through pores, if thetransverse dimension of the pores were not very muchgreater than its molecular dimension.

For measuring the pre- and post-ischemic K s for BBBi

permeation of sucrose and inulin we employed the graphi-w xcal method of Patlak et al. 12 in which values for the

distribution space of a tracer are obtained at different timesafter its i.v. injection, and K is obtained basically as thei

slope of the space vs. time relationship. Distribution spaceexpands as the tracer moves out of the vascular space intoneural parenchyma. This analytical method has severaladvantages over other methods of K measurement, amongi

which are elimination of the model dependence of com-partmental analysis, elimination of a correction for, or theflushing out of, tracer in the vascular space, and theacquisition of a value, V , for the initial distribution spacei

of the tracer. Determination of V can be of significance ini

assessment of BBB transfer mechanisms, since a valueappreciably greater than the tissue volume of plasma indi-

Ž .cates a non-vascular component s of the initial distribu-tion of tracer. This could be a real space that is in rapid,tracer equilibrium with plasma, or it could reflect bindingof tracer to the luminal surface of endothelial cells. Indeed,partly on the basis of V values, endothelial cell binding ofi

tracer solute followed by vesicular engulfmentŽ .adsorptive-phase endocytosis has been proposed as thepredominant mechanism for permeation of the intact BBB

w xby inulin, L-glucose, and other saccharides 9 . However,this hypothesis contends with the longer established con-

Žcept that various organic non-electrolytes except carrier-.transported ones, e.g., D-glucose traverse the intact BBB

predominantly by dissolution in and diffusion through thelipid bilayer of endothelial cell plasma membranesw x4,5,8,11,17 .

Although the present study was not aimed at assessmentof solute transfer mechanisms in the intact BBB, the

Ž .results obtained with the non-ischemic control rats werenot consistent with trans-barrier movement of sucrose andinulin occurring solely by dissolution-diffusion, or involv-ing an adsorptive process. Following brain ischemia, BBBpermeability to sucrose and inulin increased in a mannerindicative of barrier opening through the formation ofpores.

2. Materials and methods

2.1. Animals and tracers

Experiments were performed on 16 specific pathogen-Žfree, 342–374 g male Sprague–Dawley rats from the

.NRCC colony that had been housed singly in hangingwire cages and with unrestricted access to Purina chowŽ .No. 5001 and filtered tap water. The animals were

Ž .assigned randomly to control ns8 and ischemic injuryŽ .ns8 groups.

Fresh stocks of radiotracers were obtained a few weeksŽ .before use from New England Nuclear Boston, MA

Ž .through Du Pont Canada Mississauga, Ont. .w14 Ž .x ŽC U Sucrose catalog No. NEC100X, 475 mCirmmol,

.100 mCirml in 9:1 ethanol:water was stored at y108C;aliquots for injectates were subjected to a gentle air stream

w3 xto evaporate the ethanol before mixing with H inulin andŽ .saline for injection. Weighed portions ca. 2 mg of

w3 Ž .x ŽH G inulin catalog No. NET314L, 212 mCirg, crys-.talline solid were dissolved in 2 ml of ultrapure water and

dialyzed for 16 h in SpectrarPor w tubing with a molecularŽ .mass cutoff of 3500 Da Spectrum Medical, Houston, TX

Ž .against 1 l of ultrapure water changed after 8 h to whicha drop of chloroform had been added to inhibit bacterial

w3 xgrowth. Dialyzed solutions of the H inulin were stored at48C and used within 1 week of preparation. The radioactiv-ity retained by the dialysis tubing was 70–75% of initialradioactivity.

2.2. Ischemic opening of the BBB

Forebrain ischemia was produced by an adaptation ofw xthe 2VO method of Smith et al. 20 . Each rat was

Ž .anesthetized with sodium pentobarbital 65 mgrkg, i.p. ,Žintubated, and mechanically ventilated Harvard rodent.ventilator, Ealing Scientific, St. Laurent, PQ with a 30:70

mixture of O and N . Tympanic and colonic temperatures2 2

( )E. Preston, D.O. FosterrBrain Research 761 1997 4–106

were measured using YSI 511 and 402 thermistor probesŽ .Yellow Springs Instruments, Yellow Springs, OH and apolygraph recorder, and both temperatures were main-tained between 37.5 and 38.08C by means of a molded,circulating water pad on which the animal rested. The tailartery was cannulated, blood gases and pH were moni-tored, and ventilation was adjusted, if required, to obtainpCO and pO within the normal ranges. The common2 2

carotid arteries were exposed and freed for later placementof occlusive vascular clamps. Arterial pressure was low-ered by withdrawing blood from the tail artery into aheparinized syringe; it was maintained at 42–47 mmHgŽmonitored by a pressure transducer and the polygraph

.recorder by adjustment of the volume of blood in thesyringe. Both carotid arteries were then occluded for 10min, after which the withdrawn blood was returned to therat. The incisions were closed with sutures, mechanicalventilation was withdrawn, and the rat was allowed torecover in a heat lamp-warmed, plastic cage containingbedding, food and water.

2.3. Measurement of K s and V s for brain uptake ofi i

sucrose and inulin

These measurements were made 6 h after the ischemicinsult, and also in control, non-ischemic rats. The latterwere not subjected to sham 2VO ischemia, since in a

w xprevious study 14 a sham procedure of hypovolemichypotension without carotid occlusion had no significanteffect on BBB permeability to sucrose. Each animal was

Žanesthetized with sodium pentobarbital i.p. controls: 65mgrkg; previously anesthetized, post-ischemic rats: 45

.mgrkg and cannulas were placed in a femoral artery andvein. Body temperature was measured with a colonic probeand maintained, as above, at 37.5–38.08C. The rat was

Ž .heparinized 300 unitsr0.3 ml, i.v. and then given an i.v.bolus injection of 0.5 ml of isotonic NaCl solution contain-

w14 x Ž . w3 x Žing C sucrose 4 mCir100 g and H inulin 12.mCir100 g . The tracers were allowed to circulate for 3,

10, 20, or 30 min at which point the rat was killed bydecapitation and a blood sample was collected immedi-ately from the severed neck arteries for determination ofthe final plasma concentration of each tracer. Throughoutthe period of tracer circulation arterial blood was with-drawn by syringe pump at a constant rate set to obtain asample of 0.5 ml in a 0.5 ml insulin syringe with low dead

Ž .space U100; Becton Dickinson, Franklin Lakes, NJ . Thebrain was immediately removed, placed on moistened filterpaper on a chilled plate, stripped of meningeal andchoroidal membranes and vessels, and divided intoanatomic regions, which were placed in tared scintillationvials and weighed. These tissue samples and three 50 mlaliquots of plasma from each blood sample were digested

w Žwith 1.5 ml of Soluene 350 Packard Instrument, Gronin-.gen, Netherlands at 378C for 24 h. Ten ml of Cytoscint

w Ž .ES ICN Biomedicals, Costa Mesa, CA was added to

each vial, and after allowing for decay of chemilumines-cence, radioactivity was measured in a Beckman 6800liquid scintillation spectrometer calibrated to report thedpm of each isotope.

For each rat and each radiotracer the following valueswere obtained: C , the regional tissue concentration ofbrain

Ž .tracer dpmrg wet tissue ; C , the final plasmafinal plasma

concentration of tracer at the time of decapitation; and H t0

C d t, the integral of the plasma concentration of tracerplasmaŽ y1 .over the circulation time dpm s ml , obtained by multi-

plying the tracer concentration in the plasma of the syringeŽpump blood sample C , dpmrml; corrected for dilu-plasma

. Ž .tion by deadspace saline by the circulation time t inseconds. The data were graphed, by brain region and

Ž . ttracer, with C rC as ordinate mlrg and Hbrain final plasma 0Ž .C d trC as abscissa s . Linear regressionplasma final plasma

analysis was applied to each data set, which consisted ofeight points corresponding with the eight rats of each

Ž .group control or ischemic that were treated identicallyexcept for variation from 3 to 30 min in the time allowedfor tracer circulation. The linear regression is described by

w xthe equation after Patlak et al. 12 : C rC sKbrain final plasma iŽ t .H C d trC qV . The left side of this equa-0 plasma final plasma i

tion expresses the total tracer content of a brain tissuesample in terms of an equivalent plasma space. This totaltracer distribution space is expressed on the right side asthe sum of a parenchymal tracer space and a ‘vascular’tracer space, V , which is the apparent initial volume ofi

tracer distribution, comprising actual plasma space plusany rapidly equilibrating space, andror its equivalent dueto binding of tracer to the surface of endothelial cells.

2.4. Regression analyses and statistics

The SYSTAT w 5.0 statistical and graphical softwareŽ .program Systat, Evanston, IL was used for data analysis.

Linear regression analysis of each data set yielded theŽ . Ž .slope K , the zero-time, ordinate intercept V , and theiri i

Ž .standard errors S.E.M. . Analysis of covariance was usedto obtain P values for differences in K and V betweeni i

tracers and between control and ischemic groups, withineach brain region. For multiple comparisons Bonferroniadjustments to the probabilities were applied. The S.E.M.s

Žof differences between K s DK , ischemia – control;i i. w xsame tracer only were computed 23 with the aid of basic

statistical parameters generated by SYSTAT. For compar-w xisons between DK s Welch’s approximate t 23 was used.i

3. Results

Fig. 1 illustrates, using results for striatum, the nature ofthe data obtained and their graphical analysis. K s fori

striatal BBB permeation of sucrose and inulin, in theŽcontrols and in the BBB-injured rats 6 h after 10 min of

.forebrain ischemia , are indicated by the slopes of the

( )E. Preston, D.O. FosterrBrain Research 761 1997 4–10 7

Fig. 1. Multiple time, graphical analysis of blood-to-brain flux ofw14 x w3 xC sucrose and H inulin in striatum of rats given a combined i.v.bolus injection of the tracers. Filled symbols indicate measurements on

Ž .rats subjected to forebrain ischemia 6 h earlier ns8 ; open symbols areŽ .for control rats ns8 . Measurements were made for tracer circulation

times of 3, 10, 20, and 30 min. The scale units for the ordinates andabscissas, and the mathematical relation between them, are explained inSection 2. The lines represent the least-squares linear regressions of the

Ž .data, and their slopes give the transfer constants K s for tracer influxi

through the BBB. The ordinate intercepts give V s, the apparent initiali

volumes of tracer distribution.

regression lines, from which it is evident that for eithertreatment group permeability to sucrose was several timesgreater than permeability to inulin, and that permeability to

both tracers increased following ischemia, but more so forsucrose than inulin. As shown by the zero-time interceptsof the regression lines, the apparent initial volume of tracerdistribution, V , was approx. 4 mlrg for both sucrose andi

Ž .inulin in the controls, with small, but significant P-0.05increases being apparent following ischemia. Graphed inthe manner of Fig. 1, the data for the other brain regions

Ž .examined see below were generally of similar qualityŽ 2regarding coefficients of linear regression i.e., r values

ranged from 0.66 to 0.98, except for a value of 0.21 for.inulin in control cerebellum . Moreover, the K s obtainedi

were apparently not compromised by back diffusion oftracer, since, as in Fig. 1, all data sets indicated linearity oftracer uptake over the 3–30 min range of circulation timesused.

For all six brain regions examined in both the controland post-ischemic rats the K s for inulin were significantlyi

Ž . Ž .lower P-0.01 than those for sucrose Table 1 , indicat-ing a lower rate of BBB passage for the larger tracer. Mostregions exhibited significant increases in the K s for bothi

tracers following 2VO ischemia, with the most markedincreases occurring in striatum and hippocampus. Irrespec-

Ž .tive of brain region the increases DK s were about 3i

Table 1Ž y1 y1. Ž .Transfer constants K s; nl g s for blood-brain barrier permeation of radiolabeled inulin and sucrose in control rats ns8 and in rats subjected toi

Ž .10 min forebrain ischemia 6 h earlier ns83 14w x w xBrain region H Inulin C Sucrose

Ž . Ž . Ž . Ž . Ž . Ž .1 Control 2 Ischemia 3 DK 4 Control 5 Ischemia 6 DKi i

aFrontal cortex 0.30"0.02 0.45"0.05 0.14"0.05 1.90"0.25 2.31"0.12 0.41"0.28c bParietal cortex 0.18"0.04 0.98"0.13 0.80"0.13 1.71"0.27 3.61"0.55 1.90"0.61c c bStriatum 0.31"0.03 1.64"0.10 1.33"0.11 1.56"0.21 4.86"0.34 3.31"0.40c c bHippocampus 0.24"0.05 1.25"0.21 1.00"0.22 1.53"0.16 4.78"0.55 3.25"0.57c c aMidbrain 0.31"0.09 1.17"0.11 0.86"0.15 1.91"0.29 4.24"0.36 2.32"0.46

aCerebellum 0.26"0.20 0.61"0.12 0.36"0.23 1.65"0.32 2.90"0.28 1.24"0.43

Ž .Values in columns 1, 2, 4 and 5 are the slopes linear regression coefficients "S.E.M. obtained in the multiple time, graphical method of K iŽ .measurement. Values in columns 3 and 6 are the differences ischemiaycontrol between slopes"S.E.M. of the difference.

a,b,c P-0.05, P-0.01, P-0.001, respectively, for comparisons within brain region and tracer, between control and ischemia, and within brain regionŽ .between DK s. All comparisons within brain region between values in columns 1 and 4 and between values in columns 2 and 5 demonstrated highlyi

Ž . Ž .significant differences P-0.01 . All regression coefficients, except that for cerebellum in column 1, were significantly different P-0.01 from zero.

Table 2Ž y1 . Ž .Volumes V s; ml g of plasma plus rapidly equilibrating tracer space in brain regions of control rats ns8 and of rats subjected to 10 min forebraini

Ž .ischemia 6 h earlier ns83 14w x w xBrain region H Inulin C Sucrose

Control Ischemia Control IschemiacFrontal cortex 5.8"0.1 6.0"0.2 6.3"0.7 7.4"0.3

Parietal cortex 5.4"0.1 6.0"0.4 6.1"0.8 8.2"1.4a a,bStriatum 4.0"0.1 4.9"0.3 3.9"0.6 7.0"0.9

Hippocampus 4.7"0.2 5.9"0.7 5.0"0.5 7.8"1.4Midbrain 6.2"0.3 5.9"0.4 6.5"0.8 7.0"0.9Cerebellum 8.9"0.7 9.2"0.4 11.1"0.9 10.5"0.7

Values were obtained as the Y intercepts"S.E.M. of linear regressions applied in the multiple time, graphical method of measuring transfer constantsŽ .K s .ia P-0.05 for comparison within brain region and tracer, between ischemia and control.b,c P-0.05 and P-0.01, respectively, for comparison within brain region and ischemia, between sucrose and inulin.

( )E. Preston, D.O. FosterrBrain Research 761 1997 4–108

Table 3Ž .Ratios of transfer constants from Table 1 for blood-brain barrier perme-

ation of sucrose and inulin in control and post-ischemic rats

Brain region DK sucroserK sucroserK inulin ii i

DK inuliniControl Ischemia

Frontal cortex 6.2 5.2 2.9Parietal cortex 9.3 3.7 2.4Striatum 5.1 3.0 2.5Hippocampus 6.3 3.8 3.2Midbrain 6.2 3.6 2.7Cerebellum 6.5 4.7 3.5

a a,b aMean"S.E.M. 6.6"0.6 4.0"0.3 2.9"0.2

a Ž .Significantly different from unity P -0.001 .b Ž .Significantly different from mean value for controls P -0.01 .

times greater for sucrose than inulin, although relative tobaseline values the percentage or fold increases weregreater for inulin.

For the controls there were no significant differencesŽ .between regional V s for sucrose and inulin Table 2 . Ini

the post-ischemic rats there was a trend for sucrose andinulin V s to be somewhat higher than the control values,i

Ž .but the difference was significant P-0.05 only in stria-tum.

Table 3 presents comparisons of the relative magnitudesŽ .i.e., ratios of the sucrose and inulin K s and DK si i

reported in Table 1. Across the six brain regions examinedthe sucroserinulin K ratios were markedly similar withini

each treatment group, averaging to 6.6 in the control ratsand to a significantly lower value of 4.0 in the post-ischemic rats. The sucroserinulin DK ratios were alsoi

very similar across the regions, with an average value of2.9. As discussed below, these ratios help to characterizethe mechanisms underlying BBB passage of the tracers,particularly those involved in ischemic opening of thebarrier.

4. Discussion

K , the unidirectional transfer constant for blood-to-braini

transfer of a solute across the BBB, is virtually equal toPS, the BBB permeability-surface area product, when thecapillary extraction fraction of the solute is very low, as is

w xthe case for both sucrose and inulin 5 . However, in thefollowing discussion, use of the term PS rather than K isi

sometimes necessary for correct expression of certain rela-tionships.

4.1. Tracer permeation of the intact BBB

The ways in which sucrose and inulin permeate theintact BBB have not been established unequivocally. Com-parative analyses of the blood-to-brain movement of vari-

Ž . Žous small MW-400 , organic non-electrolytes including.sucrose , for which carrier-mediated transport does not

occur, have indicated that permeation is proportional to theŽ w x.solute’s lipid solubility and diffusivity reviewed in 5 . It

is inferred, therefore, that such solutes permeate the BBBprincipally by dissolving in and diffusing through the lipid

Žbilayer of endothelial cell plasma membranes plus diffus-.ing through the cytoplasm of the endothelial cells .

An expression that describes quite well the permeabil-ity-solubility-diffusivity relationship for organic non-elec-trolytes is PSAPC rMW0.5, where PC is theOrW OrW

octanolrwater partition coefficient of the solute, and0.5 w xMW is the square root of its molecular weight 5 .

Ž .PC is used to approximate the membrane lipid rwaterOrW

partition coefficient of the solute. MW 0.5 is used as anŽindex of the molecular radius of the solute Stokes–Ein-

.stein r ; it appears in the denominator of the expression,Žbecause diffusivity normally expressed as a diffusion

.coefficient, D is inversely proportional to r. The PCOrWŽ . y4 w xfor sucrose MW s 342 is 2.14 = 10 7 ; thus

PC rMW0.5 is 1.16=10y5. The PC for inulinOrW OrWŽ . y4 w xMWf5000 is 0.26=10 as calculated 7,9 from its

w x 0.5 y7butanolrwater PC 9 ; thus PC rMW is 3.68=10 .OrW

The PS rPS ratio calculated using these values andsuc inu

the above relationship is 31.5. The ratio is 23.4 if the˚ w xactual molecular radii of the tracers, 4.6 and 13.1 A 1 ,

respectively, are used instead of MW 0.5. Either estimate isseveral times greater than the mean sucroserinulin K i

Žratio of 6.6 presently obtained with the control rats Table.3 .

Ž .The regional K s for sucrose in control rats Table 1 ,i

which averaged to 1.71 nl gy1 sy1, were similar to valuesw3 xobtained with H sucrose in two recent studies from this

w xlaboratory 14,15 ; they were intermediate between valuesw x Ž y1 y1.reported by Ravnborg et al. 18 0.26–0.49 nl g s

w x Ž y1and values obtained by Ohno et al. 10 5.3–7.5 nl gy1 .s for the same brain regions. The regional K s fori

Ž .inulin in the control rats Table 1 were similar to valuesw x w xreported by Ohno et al. 10 and Ziylan et al. 24 , but only

w xabout one-tenth those obtained by Amtorp 1 and Lucch-w xesi and Gosselin 9 . The discrepancies among laboratories

in K s for sucrose and inulin impede consolidated assess-i

ment of the mechanisms of the BBB permeation of thesesubstances. However, when, as here, sucrose and inulinK s are measured simultaneously in the same animals,i

Ž .making capillary surface area S the same for each tracer,the ratio of their K s presumably reflects the relativei

Ž .permeabilities P of the BBB to them, information thatcan help in distinguishing among possible mechanisms of

w xpermeation 5,9 .If it is assumed for the moment that BBB permeation of

sucrose occurred solely by the dissolution-diffusion pro-cess, the large discrepancy between the currently observed

Ž .sucroserinulin K ratio 6.6 on average; Table 3 and thati

predicted using the relationship PS A PC rMW0.5OrW

Ž .31.5; see above suggests that permeation of inulin throughthe intact BBB occurred largely by a mechanism otherthan dissolution-diffusion. That is, permeation of inulin,

( )E. Preston, D.O. FosterrBrain Research 761 1997 4–10 9

relative to sucrose, was several times greater than wouldbe predicted for a dissolution-diffusion process. Amongmechanisms of permeation that might operate in parallel

Žwith dissolution-diffusion are micropinocytosis fluid-phase.endocytosis coupled with vesicular transport, diffusion

through aqueous pores or channels, and adsorptive-phaseendocytosis.

w xLucchesi and Gosselin 9 concluded that inulin perme-ation of the BBB occurs principally by adsorptive-phaseendocytosis. Central to this conclusion was their determi-

w3 x Ž .nation of an average regional V for H inulin 19 mlrgiw14 x Ž .that was larger than that for L- C glucose 12 mlrg and

Ž .much larger than the average plasma volume V ofpŽ .cerebral tissue 5.6 mlrg determined with 70 kDa

w14 x Ž .C dextran. The non-plasma components V yV of thei p

initial distributions of inulin and L-glucose were attributedŽ .to binding adsorption of inulin and L-glucose to the

luminal surface of endothelial cells. In the present study,however, the regional V s for inulin in control rats werei

Ž .not significantly different from sucrose V s Table 2 ,iw xwhich is in agreement with results of Smith et al. 21 , and

they were also closely comparable with regional plasmaŽ .volumes V s determined with high molecular weightpw x w xdextrans 9,21 or radioiodinated albumin 13 . Thus, our

data provided no indication of appreciable binding ofinulin to endothelial cells, or support for the hypothesisthat adsorptive-phase endocytosis largely accounts for themovement of inulin across the intact BBB.

The operation of fluid-phase endocytosis coupled withvesicular transport, or of diffusion through aqueousporesrchannels, or of both processes in the permeation ofthe intact BBB by inulin presumably would mean the

Ž .operation of the process es also in the permeation of thesmaller molecule, sucrose. On the basis that permeation ofthe intact BBB by sucrose appears to be explicable mainly

Ž w x.by a dissolution-diffusion process see Fig. 8 of 5 theputative, additional pathway supporting inulin influx wouldbe a minor one for sucrose. As the following calculationsindicate, this condition can be met by a vesicular model.Taking, for example, the control data for frontal cortexŽ .Table 1 , and supposing that pinocytosis supplementsdissolution-diffusion, the measured K for inulin, 0.30 nli

gy1 sy1, would be the sum of two K s, one for dissolu-i� 4tion-diffusion, K inu and one for pinocytosis,i dd

� 4 � 4 � 4K inu . Thus, K inu qK inu s0.30. Simi-i pino i dd i pino� 4 � 4larly, for sucrose flux, K suc qK suc s1.90. As-i dd i pino

� 4 � 4suming that K suc s K inu and thati pino i pino� 4 � 4 Ž .K suc rK inu s31.5 see above , the values of thei dd i dd

four primary K s can be calculated. From the values thusi

obtained sucrose flux would comprise 13% pinocytosisand 87% dissolution-diffusion, while inulin flux would be83% pinocytosis and 17% dissolution-diffusion. In con-trast, analogous calculations for a model in which dissolu-tion-diffusion is supplemented by diffusion of sucrose andinulin through pores – assuming this diffusion to be in

Žproportion to the ratio of free diffusion coefficients Df,

w x.rD s2.9 1 -— would indicate 40% of sucrosesuc f, inuŽflux and 88% of inulin flux to occur via the pores. The

pore model yields greater than 40% sucrose permeation viathe pores if it is assumed that the pores are not broad

wenough to permit unhindered diffusion of both tracers seex .below . However, these calculations can only be consid-

ered as illustrative at this point, and measurements of thesimultaneous trans-barrier flux of a number of differenttracer pairings would be needed to form a hypothesis on

Ž .the nature of the putative pathway s supplementing disso-lution-diffusion. As a final point here, rejecting dissolu-tion-diffusion as the principal mechanism of sucrose per-meation of the intact BBB would make it difficult toreconcile the results with either a pore or a vesicularmodel, without postulating BBB features that are inconsis-tent with current knowledge.

4.2. Tracer permeation of the BBB after ischemic injury

w3 x w xAs previously demonstrated with H sucrose 14 ap-preciable opening of the BBB was evident 6 h after 10 min

w3 xof 2VO ischemia, with permeability to H inulin as wellw14 xas to C sucrose being increased in several brain regions,

Ž .although the absolute magnitudes of the increases DK siŽ .were less for inulin than for sucrose Table 1 .

Across brain regions, the sucroserinulin K ratio de-i

clined from a mean of 6.6 in the controls to 4.0 in theŽ .post-ischemic rats Table 3 , which suggests increased

Ž .participation post ischemia of a mechanism or mecha-nisms other than dissolution-diffusion in overall tracerflux. Characteristics of the physical nature of the mecha-

Ž .nism s are suggested by comparison of the ischemia-in-duced increments in the K s for the two tracers. Acrossi

brain regions the sucroserinulin ratios of DK s wereiŽ .markedly similar, and their average value of 2.9 Table 3

Ž .was significantly different from unity P-0.001 . ThisŽfinding is inconsistent with vesicular transport fluid-phase

.endocytosis and transcytosis as the basis of the increasesin BBB permeability to the tracers, unless the loading ofvesicles were to occur by diffusion through vesicularnecks, with discrimination between inulin and sucrosearising from the difference in their molecular dimensions.However, there being no compelling evidence for re-stricted loading of vesicles, a non-vesicular mechanism ofthe post-ischemic increases in BBB permeability to su-crose and inulin seems more likely. The finding that theaverage sucroserinulin ratio of DK s corresponded to thei

Žsucroserinulin ratio of free diffusion coefficients Df,w x.rD s2.9, water, 388C 1 strongly suggests that thesuc f, inu

BBB opening following brain ischemia involved the for-mation of trans- or paracellular aqueous pores or channelsin the capillary endothelium.

The increase in the transit of sucrose and inulin inŽ .proportion to their free diffusion coefficients D couldf

imply the formation of pores broad enough not to hinder˚Žthe diffusion of inulin Stokes–Einstein radiuss13.1 A

( )E. Preston, D.O. FosterrBrain Research 761 1997 4–1010

w x. Ž1 . This would require, according to pore theory see,w x.e.g., 3 , pores with a radius about 100 times that of inulin

Ž .to reach 0.96 D . However, pores of this size would havef

the potential for appreciable convective solute transferŽ .bulk flow , the occurrence of which, although uncertain inthe present case, would have reduced discrimination be-tween sucrose and inulin to something less than their Df

ratio. Thus, smaller pores may be more plausible. In thecase, for example, of pores with a radius 10 times that ofinulin, the diffusion of sucrose and inulin would be re-stricted to approx. 0.86 D and 0.64 D , respectivelyf fŽ w x.calculated with equations given in 3 , and the expected

Ž . Žratio of DK s sucroserinulin would be approx. 3.9 i.e.,iw x w x.0.86r0.64 D ratio , if diffusion were the only processf

of tracer movement through the pores. The addition ofconvective transfer through such pores, which would entail

w xlittle discrimination between sucrose and inulin 3 , couldhave lowered the sucroserinulin ratio to the observedaverage value of 2.9. Alternatively, the correspondencebetween the ratio of DK s and the ratio of D s might bei f

explained without inferring the existence of pores per se.Conceptually, endothelial cells with areas of severely,

Žischemically injured plasma membrane e.g., membrane.gaps, blebs, disorganized lipid bilayer could provide a

broad pathway for blood-to-brain diffusion of the tracers –their intracellular fluid. Electron microscopic examinationof the microvasculature of the most injured brain regionsŽ .striatum and hippocampus will likely be necessary toadvance characterization of the pore-like opening of theBBB effected by forebrain ischemia in the rat.

Acknowledgements

The authors thank Ms. Patricia Mills for expert techni-cal assistance in performing surgeries, radiotracer measure-ments, and data analysis.

References

w x1 O. Amtorp, Estimation of capillary permeability of inulin, sucroseŽ .and mannitol in rat brain cortex, Acta Physiol. Scand. 110 1980

337–342.w x2 J. Dobbin, H.A. Crockard, R. Ross-Russel, Transient blood-brain

barrier permeability following profound temporary global ischemia:w14 xan experimental study using C AIB, J. Cereb. Blood Flow Metab.

Ž .9 1989 71–78.w x3 F.-R.E. Curry, Mechanics and thermodynamics of transcapillary

Ž .exchange, in: E.M. Renkin, C.C. Michel Eds. , The CardiovascularSystem, vol. IV, Microcirculation, American Physiological Society,Bethesda, MD, 1984, pp. 309–374.

w x4 J.D. Fenstermacher, Current models of blood-brain transfer, TrendsŽ .Neurosci. 8 1985 449–453.

w x5 J.D. Fenstermacher, S.I. Rapoport, Blood-brain barrier, in: E.M.Ž .Renkin, C.C. Michel Eds. , The Cardiovascular System, vol. IV,

Microcirculation, American Physiological Society, Bethesda, MD,1984, pp. 969–1000.

w x6 T. Kirino, Delayed neuronal death in the gerbil hippocampus follow-Ž .ing ischemia, Brain Res. 237 1982 57–69.

w x7 A. Leo, C. Hansch, D. Elkins, Partition coefficients and their uses,Ž .Chem. Rev. 71 1971 525–616.

w x8 V.A. Levin, Relationship of octanolrwater partition coefficient andmolecular weight to rat brain capillary permeability, J. Med. Chem.

Ž .23 1980 682–684.w x9 K.J. Lucchesi, R.E. Gosselin, Mechanism of L-glucose, raffinose,

and inulin transport across intact blood-brain barriers, Am. J. Phys-Ž .iol. 258 1990 H695–H705.

w x10 K. Ohno, K.D. Pettigrew, S.I. Rapoport, Lower limits of cerebrovas-cular permeability to nonelectrolytes in the conscious rat, Am. J.

Ž .Physiol. 235 1978 H299–H307.w x11 W.H. Oldendorf, Lipid solubility and drug penetration of the blood

Ž .brain barrier, Proc. Soc. Exp. Biol. Med. 147 1974 813–816.w x12 C.S. Patlak, R.G. Blasberg, J.D. Fenstermacher, Graphical evalua-

tion of blood-to-brain transfer constants from multiple-time uptakeŽ .data, J. Cereb. Blood Flow Metab. 3 1983 1–7.

w x13 E. Preston, N. Haas, Defining the lower limits of blood-brain barrierpermeability: factors affecting the magnitude and interpretation of

Ž .permeability-area products, J. Neurosci. Res. 16 1986 709–719.w x14 E. Preston, J. Saunders, N. Haas, M. Rydzy, P. Kozlowski, Selec-

tive, delayed increase in transfer constants for cerebrovascular per-meation of blood-borne 3H-sucrose following forebrain ischaemia in

Ž . Ž .the rat, Acta Neurochir. 51 Suppl. 1990 174–176.w x15 E. Preston, G. Sutherland, A. Finsten, Three openings of the blood-

brain barrier produced by forebrain ischemia in the rat, Neurosci.Ž .Lett. 149 1993 75–78.

w x16 W.A. Pulsinelli, J. Brierley, F. Plum, Temporal profile of neuronaldamage in a model of transient forebrain ischemia, Ann. Neurol. 11Ž .1982 491–498.

w x17 S.I. Rapoport, K. Ohno, K.D. Pettigrew, Drug entry into brain, BrainŽ .Res. 172 1979 354–359.

w x18 M. Ravnborg, G.M. Knudsen, M. Blinkenberg, No effect of pulsedmagnetic stimulation on the blood-brain barrier in rats, Neuroscience

Ž .38 1990 277–280.w x19 S. Sasaki, R. Ferszt, J. Cervos Navarro, Transendothelial vesicular´

transport of protein in brain edema induced by ultraviolet irradiation,Ž .Acta Neuropathol. 40 1977 207–212.

w x20 M.L. Smith, R.N. Auer, B.K. Seisjo, The density and distribution ofischemic brain injury in the rat following 2–10 min forebrain

Ž .ischemia, Acta Neuropathol. 64 1984 319–332.w x21 Q.R. Smith, Y.Z. Ziylan, P.J. Robinson, S.I. Rapoport, Kinetics and

distribution volumes for tracers of different sizes in the brain plasmaŽ .space, Brain Res. 462 1988 1–9.

w x22 J.C. Villegas, R.D. Broadwell, Transcytosis of protein through them am m alian cerebral epithelium and endotheliumII. Adsorptive transcytosis of WGA-HRP and the blood-brain and

Ž .brain-blood barriers, J. Neurocytol. 22 1993 67–80.w x23 J.H. Zar, Biostatistical Analysis, Prentice-Hall, Englewood Cliffs,

NJ, 1984.w x24 Y.Z. Ziylan, P.J. Robinson, S.I. Rapoport, Differential blood-brain

w14 x w3 xbarrier permeabilities to C sucrose and H inulin after osmoticŽ .opening in the rat, Exp. Neurol. 79 1983 845–857.