Bram Research, 550 (1991) 257-262 © 1991 Elsevier Science Pubhshers B V 0006-8993/91/$03 50 ADONIS 000689939116629Y
257
BRES 16629
Oleic acid reversibly opens the blood-brain barrier Ldszl6 Sztrlha 1'4 and A. Lorrls
B e t z 1-3
Departments of ;Surgery, 2Pediatricsand 3Neurology, Umverstty of Michigan, Ann Arbor, MI 48109 (U S A ) and 4Department of Pediatrics, Szent-Gyorgyt Medical School, Szeged (Hungary) (Accepted 18 December 1990)
Key words Olelc acid, Blood-brain barner, a-Ammolsobutync acid permeablhty surface area product, Permeabdlty, Barrier modlficaUon
This study examined the effect of mtracarotld olelc acid infusion on blood-brain barrier permeabdlty Oleic acid was infused for 30 s at a rate of 6 ml/mm into the right internal carotid artery at concentrations of 10-6, 10-5, 2 x 10-5 and 5 x 10-5 M Extensive Evans blue-albumin extravasatlon was observed 15 mm after the administration of 2 x 10-5 M olelc acid The permeabdlty surface area product for a-ammolsobutync aod (AIB), determined 1-11 mln following the infusion of olelc acid was increased 10-fold following infusion of 10-5 M olelc acid and 20-fold following the administration of 5 × 10-5 M oleate The blood-brain barrier opemng to AIB proved to be reversible 80-90 mm after the infusion of 2 × 10-5 M olelc acid The possible mechamsms of the olelc acid effect are discussed INTRODUCTION
The capillaries in brain are formed by a speclahzed endothehum whose function ~s to regulate the movement of solutes between blood and brain The endothelial cells in brain capillaries are sealed together by continuous tight junctions and contain few pmcytottc vesicles These properties hmlt the free exchange of water-soluble non-electrolytes, electrolytes and proteins from blood to brain and are responsible for formation of a blood-brain burner (BBB) 6'25 27 Since brain uptake of many neuropharmacologlc agents is limited by the BBB, a method for reversibly increasing cerebrovascular permeabdity could enhance treatment of a variety of neurologlc d~seases Osmotic BBB opening has been characterized quantitatively and shown to be reproduoble and reversible 23 It is believed to increase BBB permeabdlty by disrupting mtracellular tight junctions 4 Chmcally, this procedure has been shown to increase the delivery of chemotherapeutic agents to the brain I7, but its use for treatment of patients is not yet widely accepted 57 Alternative approaches to increasing BBB permeabdity would be to alter endothehal cell membrane integrity or to actwate mtracellular second messenger systems that regulate BBB permeabdlty In recent years, there have been many studies on the biological effects of the fatty acids ~ 11 1~,i4 18,2029 and there is growing evidence that they modify membrane structure and function 26 Low concentrations of several unsaturated fatty acids such as
olelc, hnolelc and hnolemc acid exert a stabshzlng effect on the erythrocyte membrane, whereas higher concentrations of these same agents cause membrane lysls 26 Furthermore, protein kmase C, an enzyme which can be detected in brain capillaries 2z and plays an important role in the regulation of cellular functions 16, is activated by unsaturated fatty a c i d s 13A4 Liberation of various free fatty acids from brain tissue has been observed during injuries that are associated with BBB opening, such as ischemla ~5 and cold mjury I Much attention has been focused on the role of arachldonlc acid and less on the other unsaturated fatty acids as possible contributing agents in the BBB destruction and brain edema formation that accompanies these injuries 3'z9 In this study, we determined the effect of an lntracarot~d infusion of sodium oleate on BBB permeabdlty
MATERIALS AND METHODS Adult male Sprague-Dawley rats, weighing between 350 and 400 g were anesthetized by intramuscular rejection of 50 mg/kg ketamme hydrochlonde and 10 mg/kg xylazme Polyethylene catheters (PE50) filled with hepanmzed sahne were inserted into the right femoral vein for the admlmstration of the tracers and into the femoral arteries for continuous pressure recording and blood samphng After exposure of the right carotid artery, the occipital, superior thyroid and pterygopalatme arteries were hgated, and the right external carotid artery was catheterized for retrograde infusion Olelc acid sodium salt (Sigma, St Louis, MO) was dissolved in 20% ethanol and diluted in saline to concentrations of 10-6 , 10-5 , 2 × 10-s and 5 x 10-5 M The stock solutions were prepared at various concentrations so that the final amount of ethanol was the same m each dilution (0 004% ethanol) The osmolahty of the
Correspondence A L Betz, D3227 Medical Professional Building, Umverslty of Michigan, Ann Arbor, MI 48109-0718, U S A
258 solutions was between 270 and 290 mOsm and the pH was adjusted to 7 4 with sodium hydroxide The Olelc aod was infused into the right internal carotid artery at a rate of 6 ml/mm for ~l) s This rats of infusion visibly cleared the internal carotid artcr,~ ol blood The control rats were Infused w~th saline, pH 7 4, containing the same amount of ethanol as the olelc acid solutions A 2 ml/kg b wt dose of Evans blue (29~ g/vol) solution was injected intravenously ,n a group of 5 rats, 30 mm before the mtracarotld infusion of 2 × 10 ~ M olelC acid The animals were decapitated 15 rain after the carotid infusion and brains were removed, sliced and examined visually for Evans blue cxtravasatlon This quantity of dye binds almost completely to plasma albumin and as a dye-protein complex is a quahtatlve marker of BBB Integrity24 The permeability of cerebral blood vessels to [~H]ammolsobutyrlc acid (AIB) 2 was examined by a modification of the method of Ohno et al ~" as described previously p- A 0 1 ml saline bolus containing 45 ~Cl [3H]AIB (DuPont/NEN, Boston, MA) was injected into the femoral vein and allowed to circulate for 10 mln During this time, blood was continuously withdrawn from a femoral artery into polyethylene tubing At the end of the 10 mln circulatmn time, a blood sample was taken from the other femoral artery cannula to determine the final plasma isotope concentrations and hematocrlt The animal was then decapitated and the brain removed The brain was dissected into right and left halves and then the anterior and posterior cortex, hlppocampus, basal ganglia, cerebellar hemispheres and bralnstem were separated These tissues were dissolved in Protosol (DuPont/NEN, Boston, MA) Ahquots of plasma from the terminal blood sample were dissolved in Protosol and ahquots of the continuously withdrawn blood sample were dissolved in Protosol ethanol (2 1 v/v) Samples were counted m a two-channel hquld scintillation counter For [~H]AIB, brain uptake can be expressed as the permeability surface area (PS) product according to
d o s e s ot olelc acid
for
up
to
2 h without
obvlou,,
detrimental effects T h e m o s t p r o n o u n c e d E v a n s b l u e e x t r a v a s a t m n totl o w i n g i n f u s i o n at 2 x
10 s M oletc acid (Fig
1) was
o b s e r v e d m t h e right c o r t i c a l a r e a s a n d h l p p o c a m p u s and t h e left p a r a s a g i t t a l cortical a r e a
Occasionally Evans
blue s t a i n i n g a p p e a r e d in t h e right c e r e b e l l a r h e m i s p h e r e W i t h i n a g i v e n b r a i n r e g i o n , t h e p a t t e r n o t E v a n s blue e x t r a v a s a t l o n s h o w e d a s o m e w h a t h e t e r o g e n e o u s distribution
PS = C d f C ~ dt where C~ is the amount of extravascular tracer in the bram and Ca is the arterial plasma concentration The integral of (7. with time was calculated from the amount of isotope in the continuously withdrawn arterial blood sample (Co), the withdrawal rate (Fo) and the arterial hematocrit (Hct)
.fC~ at = co(1 - Hct)/Fo The amount of extravascular tracer tn the brain, C~v, was calculated from the total brain radmactlvity minus the terminal plasma concentration of [3H]AIB multiplied by the plasma volume For the determination of the regional plasma volumes, 15 /aCI [14C]dextran (DuPont/NEN. Boston, MA) was injected via the femoral vein 3 mln before the animal was decapitated The plasma volume of the brain (ml/g) is equal to the ratio of the 14C-counts in brain (per g) and plasma (per ml) The [3H]AIB was given 1 rain after completion of the carotid Infusion of 10-6, 10 -s, 2 × 10-s, 5 × 10-s M OlelC acid One group of rats that received 2 × 10 s M olelc acid was Injected with [3H]AIB 80 rain after the carotid infusion to examine the reverslblhty of the blood-brain barrier opening Five rats were used in the control and each experimental group The analysis of variance employing Dunnet's test was used for the statistical comparisons of experimental and control groups
RESULTS T h e p h y s i o l o g i c a l p a r a m e t e r s a r e s u m m a r i z e d In T a b l e I
T h e r e were no differences a m o n g the groups m the
mean
arterial
blood
pressure
(MABP),
arterial
pH,
p C O 2, p O 2 o r h e m a t o c r i t A n i m a l s s u r v i v e d t h e h i g h e r
Fig l Evans blue extravasatlon 15 min after right sided intracaroUd infusion of 2 × 10-5 M oleic acid The appearance of the Evans blue staining is heterogeneous, with pronounced staining In the right cortical and hIppocampal areas and the left parasagmal cortical areas
259
11t
• ACR
+
• PCR +
A
•
e-
HR
E O) ..... -¢
60'
+,
[ ] CER
I--
o a O Ix t,/'}
40
•
ACL
•
PCL
•
HL
20
[] CEL • Bs
_ , _
Control
10-6 M
5x10-5 M
10-5 M
Fig 2 Effect of different concentrations of olelc acmdon BBB permeablhty to AIB Between ] and 11 man following the infusion of 10-s M oleic a o d there was a 10-fold increase m the AIB PS product of the infused hemisphere After 5 x 10-s M oleate, a further increase was observed A C R , PCR, HR, BGR, C E R right sided anterior and posterior cortex, hmppocampus, basal gangha and cerebellum, respectwely ACL, PCL, HL, BGL, CEL similar but on the left smde BS bramstem Data points represent the means + S E M of 5 rats in each group *P < 0 05, **P < 0 01 compared with the controls and 10-6 M oleate +P < 0 05, ++P < 0 01 compared with 10-5 M oleate
The [3H]AIB PS product vaned between 2 and 4 /A/g/mm in the brain regions of the sahne-perfused control rats (Fig 2) These values are stmdar to those reported previously for normal rat brain z2 and indicate that the sudden perfuslon of the rat brain with sahne does not, by itself, cause the BBB to open No change was
observed after infusion of 10 -6 M olelc acid, but a slgmficant, 10-fold increase was measured m the areas of the right hemisphere after the admlmstraUon of 10-s M olelc acid (Fig 2) A further two-fold mcrease m the [3H]AIB PS product in the nght sided brain regions and a significant mcrease m the nght cerebellar hemisphere
40
• ACR • "
PCR
[] HR
30
[]
BGR
[]
CER
0
•
ACL
¢3 O n-
•
PCL
[]
HL
[]
BGL
[]
CEL
E
I'-
(/1
20"
10. +
÷+
o
~ Control
m 2x10-5 M 1-11 mm
BS
2x10-5 M 80-90 mln
Fig 3 Reverslbd~ty of the changes m the blood-brain barrier permeabdlty to AIB Immediately after the infusion of 2 x 10-s M olemc acid there was a large increase m the AIB PS product, however, 80-90 m m following the mfuslon the values returned to the control level Data points represent the means + S E M of 5 rats m each group *P < 0 05 compared with the controls +P < 0 05 compared with 2 x 10-5 M 1-11 mm group There was no slgmficant difference between the control and the 2 x 10-s M 80-90 m m groups
260 TABLE I Phystologtcal parameters
Values are means ± S E M of 5 animals
MABP (mm Hg) Temp (°C) ArterlalpH p~CO2 (mm Hg) PaO2 (mm Hg) Hematocrlt (%)
Control~
10 6M
10 5 M
2xlO
97 ± 6 365+01 7 37 + 0 01 45 ± 1 77 ± 3 36 ± 1
87 ± 5 367±02 7 35 + 0 01 46 + 4 73 ± 3 37 ± 1
90 ± 4 365+01 7 35 ± 0 02 38 + 5 72 ± 7 33 _+2
87 ± 5 367±01 7 36± 0 02 43 ± 3 84 ± 5 36 ± 2
and the left corttcal areas was observed after Infusion of
SM
5×10 SM
2xlO~M+ 80 m m
91 ± 5 365±01 7 31 ± 0 02 41 ± 7 72 ± 5 34 _+4
92 _+6 36 8 ± 0 ! 7 34±0 01 46 _+2 80 ± 5 36 ± 2
DISCUSSION
5 × 10-5 M olelc acid (Fig 2) The reversibility of the oleic acid-induced BBB openlng to [3H]AIB was examined 1-11 and 80-90 mln following the mfuston of 2 × 10-5 M oletc actd Thts dose caused an lntttal Increase In the A I B permeablhty of the same magnitude as a dose of 10-5 M, however, by 80-90 m m later, the permeabthty returned to the control values (Fig 3) A higher [a4C]dextran space was measured in the right stded cerebral regtons parallel wtth the mcreased A I B permeablhty, an Increase that was statistically stgnificant in the animals perfused with 5 x 10-5 M olelc acid (Table |I) This increase most likely represents extravasatton of
Olelc acid increases BBB permeabthty m a dosed e p e n d e n t , reversible m a n n e r The mcrease m the A I B PS product in the cortical regions of the left hemisphere and right cerebellar hemisphere can be explained as a result of cross perfuston to the contralateral hemisphere and tpsllateral cerebellar areas as lndtcated by the pattern of the extravasated Evans blue The heterogeneous Evans blue staining indicates an u n e v e n distribution of areas with Increased a l b u m i n p e r m e a b l h t y As the flow rate of the Infusion was high, no streaming p h e n o m e n a can be expected 28, and the cause of the u n e v e n appearance of the a l b u m m staining remains to be explamed
[a4c]dextran as a result of the extenstve BBB opening rather than a true increase In plasma volume Conse-
Smce the BBB is created by the plasma m e m b r a n e and ttght junctions of the endothelial cells, the change m BBB
quently, the values of A I B permeabthty at the higher
permeabthty caused by olelc acid might be explained as
doses of olelc a o d are probably underestimates of the true permeablhty
a h p l d - m e m b r a n e mteractlon The experiments of Raz and Llvne 26 d e m o n s t r a t e d that the osmotic fraglhty of erythrocytes is affected by olelc a o d Low concentrations
TABLE II Effect o f oletc acM on [14C]dextran space
Values are means ± S E M of 5 animals and expressed m units of/~l/g ACR, PCR, HR, BGR, CER right sided anterior and posterior cortex, htppocampus, basal gangha and cerebellum, respectJvely ACL, PCL, HL, BGL, CEL slmdar but on the left slde BS bramstem
ACR PCR HR BGR CER ACL PCL HL BGL CEL BS
Contro~
10 ~ M
IO- S M
2 x lO-S M
5 x 105M
2 × 10 ~ M + 80-~ mm
69±05 70±05 70±08 73±09 124±14 73±05 78±07 87±06 71±04 118±08 94±09
69±05 70±05 82±10 80±04 121±21 75±02 87±06 81±10 84±07 132±12 90±06
130±20 142±21 132±13 123±12 131±11 83±09 94±11 94±13 89±04 125±16 111±04
119±11 147±07 146±13 126±03 134±13 90±06 92±06 91±15 83±06 124±09 148±40
272±76* 307±77* 352±101" 279±70* 173±26"* 173±56"* 133±45 104±30 99±25 134±04 97±07
83±14 74±09 76±06 76±03 98±06 66±02 85±07 68±04 77±05 107±06 89±04
*P < 0 01 compared with the controls and the 10-s M group **P < 0 05 compared with the controls and the 10 s M group
261 reduced the proportion of cells that were lysed while higher olelc acid concentrations (10-5 M and higher) increased their susceptlblhty to osmotic shock In our experiments the same threshold concentration range of olelc acid was found to be damaging to the BBB Olelc acid IS an amphlphlhc substance which can induce major changes In membrane function 1° At low concentrations, as a monomer, ~t inserts itself into the lipid membrane, changing the physical properties and physiological function of the membrane m a way that appears as a membrane-stablhzmg effect ~° At high concentrations, however, amphlphdlc monomers aggregate into mlcelles which have the ablhty to incorporate membrane hplds into their structure The incorporation of high concentrations of amphlphlles into membranes physically &srupts the hpld bllayer and, consequently, destroys the integrity of biological membranes 1° If an olelc acidmembrane interaction is responsible for the increased permeability of the BBB, It is surprising that the process is so rapidly reversible Olelc acid is also known to be a potent actwator of protein klnase C 13'14, a key enzyme in mediating transmembrane signahng 16 The presence of protein klnases in the brain capillaries has been shown previously22 and modulation of the kinetics of protein phosphorylatlon by second messengers may contribute to the regulation of the BBB permeablhty 2~ Although there is only indirect ewdence that protein klnase C may play a role m the regulation of the BBB transport processes 9, the possibility exists that ole~c acid may affect the permeablhty of the brain vascular endothelial cells by changing protein klnase C-induced protein phosphorylatlon The rapid reverslbdlty of the olelc acid-reduced BBB opening supports the view that a reversible biochemical process ~s the underlying event Recent stu&es 1~ 18 20 have revealed yet other biological effects that are exerted by olelc acid that could influence the permeabdlty of the BBB Reduced synaptosomal Na+,K+-ATPase actlwty 2° and inhibition of the mltochondrml oxidative phosphorylatlon 19 were observed after treatment with ole~c acid The release of reactwe oxygen metabohtes may also be revolved m barrier
destruction as olelc acid-induced pulmonary edema can be attenuated by pretreatment with catalase and superoxide dlsmutase 11 Olelc a o d in cortical superfusion experiments performed by Unterberg et al 29 was found to be without effect on the BBB of pml vessels Given lntracerebrally it did not induce changes in brain water and cation levels 3 The discrepancy between our results and those of others may be explained by the different route of administration of the olelc acid Increased olelc acid concentrations were measured in the plasma and interstmally drained brain edema fluid in cats exposed to cold InJury I and in the brain tissue following lschemla s'~5 30 Our results Indicate that the elevated olelc acid concentration might contribute to the &sturbance of BBB permeablhty observed in these experimental models However, binding of olelc acid to albumin would normally reduce the concentration of free olelc acid m the plasma by a considerable amount In our studies, the lntracarotld infusion rate was high enough to completely clear the blood from the carotid circulation on the side of the infusion Thus, there was no binding of olelc acid to albumin during the infusion and the BBB was exposed to the stated concentrations of free olelc acid We are unable to predict whether albumin-bound olelc acid would have a similar effect In conclusion, mtracarotld infusion of olelc acid increases BBB permeablhty m a dose-dependent and reversible fashion The procedure is well tolerated in the short term, however, neither long-term toxicity nor localized effects leading to cellular injury, brain edema, and metabolic disturbances have been examined It is unclear at the present time whether the BBB opening reduced by oleic acid results from a non-specific lipidmembrane interaction or the actwat~on of a specific biochemical pathway Further studies are warranted since this procedure may provide a useful clinical approach to modification of BBB permeablhty
Acknowledgements The authors thank Gloria Rodnguez for her valuable secretarial support This work was supported by Grant NS 23870 from the National Institutes of Health
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mtracerebral rejection of arachldomc acid, Ann Neurol, 13 (1983) 625-632 Dorovml-Zls, K, Sato, M, Gopmg, G , Rapoport, S and Bnghtman, M, Iomc lanthanum passage across cerebral endothehum exposed to hyperosmotlcarabmose, Acta Neuropathol, 60 (1983) 49-60 Flshman, R A , Is there a therapeutic role for osmotic breaching of the blood-brain barrier9, Ann Neurol, 22 (1987) 298-299 Goldstem, G W and Betz, A L, Recent advances in understanding brain capillary function, Ann Neurol, 14 (1983) 389-395 Groothms, D R , Warkne, P C, Molnar, P, Lapin, G D and Mikhael, M A , Effect of hyperosmotlc blood-brain barrier
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disruption on transcapdlary transport m canine brain tumors J Neurosurg, 72 (1990) 441-449 Ikeda, M , Yoshlda S Busto, R Sanuso, M and Gmsberg, M D , Polyphospholnosmdes as a probable source of brain tree fatty aclds accumulated at the onset of ischemla J Neurochern 47 (1986) 123-132 Joo, F , T6sakl, A , Olah, Z, and Koltal, M , Inhlbmon by H-7 of the protein kmase C prevents formation of brain edema m Sprague-Dawley CFY rats, Brain Research, 490 (1989) 141-143 Katz, A M and Messmeo, F C , L~pld-membrane mteracuons and the pathogenesls of ~schem~c damage m the m,,ocar&um Ctrc Res, 48 (1981) 1-16 Katz, S A , Venkatachalam, M , Crouch, R K , Heftner, J E , Halushka, P V , Wise, W C and Cook, J A , Catalase pretreatment attenuates olelc acid-induced edema m isolated rabbn lung J Appl Physlol, 65 (1988) 1301-1306 Martz, D , Beer, M and Betz, A L , Dimethylthmurea reduces lschemlc brain edema wathout affecting cerebral blood flow J Cerebr Blood Flow Metab , 10 (1990) 352-357 McPhad, L C Clayton, C C and Snyderman R , A potentml second messenger role for unsaturated fatty acids actwatmn of Ca2+-dependent protein kmase, Scwnce, 224 (1984) 622-625 Murakaml, K and Routtenberg, A , Direct acuvatmn of purified protein kmase C by unsaturated fatty acids (oleate and arach~donate) m the absence of phosphollplds and C a 2+ FEBS Lett, 192 (1985) 189-193 Nakano, S , Kogure, K , Abe, K and Yae, T , Ischemla-mduced alterations m hDd metabohsm of the gerbil cerebral cortex I Changes m free fatty acid hberauon, J Neurochem 54 (1990) 1911-1916 Nestler, E J and Greengard, P , Protein phosphorylaUon in the brain, Nature, 305 (1983) 583-588 Neuwelt, E A and Barnett, P A , Blood-brain barrier disruption m the treatment of brain tumors In E A Neuwelt (Eds), lrnphcatlons o f the Blood-Bram Barrter and Its Mampulattons, Plenum, New York, 1989, pp 107-193 Ogawa, M , Yoshlda, S , Ogawa, T , Shlmada, T and Takesh~ta, M , Effect of olelc acid on mltochondrml ox~datwe phosphorylauon m rat brain shces, Btochem lnt, 17 (1988) 773-782 Ohno K Pettlgrew, K D and Rapoport, S I Lower hmas of
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cerebrovascular permeablhty to nonelectrolytcs m the consclou', rat, A m J Phy~tol, 235 (1978) H299-H307 O~shl, K , Zheng B and Kuo, J F , Inhlbmon o[ Na,K-ATPase and sodium pump by protein kmase C regulators sphmgosme lysophosphatldylchohne and oleJc acid ! Btol (hem 265 (199(I) 70-75 Olah, Z , Novak, R , Lengyel, 1 Dux E and Joo, F Kinetics of protein phosphorylauon m mlcrovessels isolated from rat brain modulation by second messengers J Neurothem 5t (1988) 49-56 Pardrldge, W M , Yang J and Elsenberg J Blood-brain barrier protein phosphorylatlon and dephosphorylatlon, J Neuro¢hem, 45 (1985) 1141-1147 Rapoport, S I Blood-Bram Barrter m Phvstolog, and Medlcme, Raven New York, 1976 Rapoport, S I Fredencks, W R , Ohno, K and Pettlgrew K D , Quantitative aspects of reversible osmotic opening of the blood-brain barrier, A m J Physlol, 238 (t980) R421-R431 Rapoport, S I , Hon M and Klatzo, I , Testing of a hypothes~s for osmouc opening of the blood-brain barrier, A m J Physlol, 223 (1973) 323-331 Raz, A and Llvne, A , Differential effects of hDds on the osmotic fragdlty of erythrocytes. Btochtm Btophys Acta 311 (1973) 222-229 Reese, T S and Karnovsky, M J . Free structural loeahzatlon of a blood-brain barrier to exogenous peroxldase J Cell Btol 34 (1967) 207-217 Sans, S C Wnght, D C . O l d f l e i d , E H andBlasberg, R G lntravascular streaming and varmble dehvery to brain following carotid artery infusions m the Sprague-Dawley rat. J Cerebr Blood Flow Metab , 8 (1988) 116-120 Unterberg A Wahl, M , Hammersen, F and Baethmann, A Permeabd~ty and vasomotor response of cerebral vessels during exposure to arachldomc acid, Acta Neuropathol, 73 (1987) 209-219 Yosh~da. S lkeda. M , Busto, R , Santiso, M , Martmez, E and Glnsberg M D , Cerebral phosphomosmde, trmcylglycerol, and energy metabohsm m reversible lschemm ongm and fate of free fatty acids, J Neurochem 47 (1986) 744-757
257
BRES 16629
Oleic acid reversibly opens the blood-brain barrier Ldszl6 Sztrlha 1'4 and A. Lorrls
B e t z 1-3
Departments of ;Surgery, 2Pediatricsand 3Neurology, Umverstty of Michigan, Ann Arbor, MI 48109 (U S A ) and 4Department of Pediatrics, Szent-Gyorgyt Medical School, Szeged (Hungary) (Accepted 18 December 1990)
Key words Olelc acid, Blood-brain barner, a-Ammolsobutync acid permeablhty surface area product, Permeabdlty, Barrier modlficaUon
This study examined the effect of mtracarotld olelc acid infusion on blood-brain barrier permeabdlty Oleic acid was infused for 30 s at a rate of 6 ml/mm into the right internal carotid artery at concentrations of 10-6, 10-5, 2 x 10-5 and 5 x 10-5 M Extensive Evans blue-albumin extravasatlon was observed 15 mm after the administration of 2 x 10-5 M olelc acid The permeabdlty surface area product for a-ammolsobutync aod (AIB), determined 1-11 mln following the infusion of olelc acid was increased 10-fold following infusion of 10-5 M olelc acid and 20-fold following the administration of 5 × 10-5 M oleate The blood-brain barrier opemng to AIB proved to be reversible 80-90 mm after the infusion of 2 × 10-5 M olelc acid The possible mechamsms of the olelc acid effect are discussed INTRODUCTION
The capillaries in brain are formed by a speclahzed endothehum whose function ~s to regulate the movement of solutes between blood and brain The endothelial cells in brain capillaries are sealed together by continuous tight junctions and contain few pmcytottc vesicles These properties hmlt the free exchange of water-soluble non-electrolytes, electrolytes and proteins from blood to brain and are responsible for formation of a blood-brain burner (BBB) 6'25 27 Since brain uptake of many neuropharmacologlc agents is limited by the BBB, a method for reversibly increasing cerebrovascular permeabdity could enhance treatment of a variety of neurologlc d~seases Osmotic BBB opening has been characterized quantitatively and shown to be reproduoble and reversible 23 It is believed to increase BBB permeabdlty by disrupting mtracellular tight junctions 4 Chmcally, this procedure has been shown to increase the delivery of chemotherapeutic agents to the brain I7, but its use for treatment of patients is not yet widely accepted 57 Alternative approaches to increasing BBB permeabdity would be to alter endothehal cell membrane integrity or to actwate mtracellular second messenger systems that regulate BBB permeabdlty In recent years, there have been many studies on the biological effects of the fatty acids ~ 11 1~,i4 18,2029 and there is growing evidence that they modify membrane structure and function 26 Low concentrations of several unsaturated fatty acids such as
olelc, hnolelc and hnolemc acid exert a stabshzlng effect on the erythrocyte membrane, whereas higher concentrations of these same agents cause membrane lysls 26 Furthermore, protein kmase C, an enzyme which can be detected in brain capillaries 2z and plays an important role in the regulation of cellular functions 16, is activated by unsaturated fatty a c i d s 13A4 Liberation of various free fatty acids from brain tissue has been observed during injuries that are associated with BBB opening, such as ischemla ~5 and cold mjury I Much attention has been focused on the role of arachldonlc acid and less on the other unsaturated fatty acids as possible contributing agents in the BBB destruction and brain edema formation that accompanies these injuries 3'z9 In this study, we determined the effect of an lntracarot~d infusion of sodium oleate on BBB permeabdlty
MATERIALS AND METHODS Adult male Sprague-Dawley rats, weighing between 350 and 400 g were anesthetized by intramuscular rejection of 50 mg/kg ketamme hydrochlonde and 10 mg/kg xylazme Polyethylene catheters (PE50) filled with hepanmzed sahne were inserted into the right femoral vein for the admlmstration of the tracers and into the femoral arteries for continuous pressure recording and blood samphng After exposure of the right carotid artery, the occipital, superior thyroid and pterygopalatme arteries were hgated, and the right external carotid artery was catheterized for retrograde infusion Olelc acid sodium salt (Sigma, St Louis, MO) was dissolved in 20% ethanol and diluted in saline to concentrations of 10-6 , 10-5 , 2 × 10-s and 5 x 10-5 M The stock solutions were prepared at various concentrations so that the final amount of ethanol was the same m each dilution (0 004% ethanol) The osmolahty of the
Correspondence A L Betz, D3227 Medical Professional Building, Umverslty of Michigan, Ann Arbor, MI 48109-0718, U S A
258 solutions was between 270 and 290 mOsm and the pH was adjusted to 7 4 with sodium hydroxide The Olelc aod was infused into the right internal carotid artery at a rate of 6 ml/mm for ~l) s This rats of infusion visibly cleared the internal carotid artcr,~ ol blood The control rats were Infused w~th saline, pH 7 4, containing the same amount of ethanol as the olelc acid solutions A 2 ml/kg b wt dose of Evans blue (29~ g/vol) solution was injected intravenously ,n a group of 5 rats, 30 mm before the mtracarotld infusion of 2 × 10 ~ M olelC acid The animals were decapitated 15 rain after the carotid infusion and brains were removed, sliced and examined visually for Evans blue cxtravasatlon This quantity of dye binds almost completely to plasma albumin and as a dye-protein complex is a quahtatlve marker of BBB Integrity24 The permeability of cerebral blood vessels to [~H]ammolsobutyrlc acid (AIB) 2 was examined by a modification of the method of Ohno et al ~" as described previously p- A 0 1 ml saline bolus containing 45 ~Cl [3H]AIB (DuPont/NEN, Boston, MA) was injected into the femoral vein and allowed to circulate for 10 mln During this time, blood was continuously withdrawn from a femoral artery into polyethylene tubing At the end of the 10 mln circulatmn time, a blood sample was taken from the other femoral artery cannula to determine the final plasma isotope concentrations and hematocrlt The animal was then decapitated and the brain removed The brain was dissected into right and left halves and then the anterior and posterior cortex, hlppocampus, basal ganglia, cerebellar hemispheres and bralnstem were separated These tissues were dissolved in Protosol (DuPont/NEN, Boston, MA) Ahquots of plasma from the terminal blood sample were dissolved in Protosol and ahquots of the continuously withdrawn blood sample were dissolved in Protosol ethanol (2 1 v/v) Samples were counted m a two-channel hquld scintillation counter For [~H]AIB, brain uptake can be expressed as the permeability surface area (PS) product according to
d o s e s ot olelc acid
for
up
to
2 h without
obvlou,,
detrimental effects T h e m o s t p r o n o u n c e d E v a n s b l u e e x t r a v a s a t m n totl o w i n g i n f u s i o n at 2 x
10 s M oletc acid (Fig
1) was
o b s e r v e d m t h e right c o r t i c a l a r e a s a n d h l p p o c a m p u s and t h e left p a r a s a g i t t a l cortical a r e a
Occasionally Evans
blue s t a i n i n g a p p e a r e d in t h e right c e r e b e l l a r h e m i s p h e r e W i t h i n a g i v e n b r a i n r e g i o n , t h e p a t t e r n o t E v a n s blue e x t r a v a s a t l o n s h o w e d a s o m e w h a t h e t e r o g e n e o u s distribution
PS = C d f C ~ dt where C~ is the amount of extravascular tracer in the bram and Ca is the arterial plasma concentration The integral of (7. with time was calculated from the amount of isotope in the continuously withdrawn arterial blood sample (Co), the withdrawal rate (Fo) and the arterial hematocrit (Hct)
.fC~ at = co(1 - Hct)/Fo The amount of extravascular tracer tn the brain, C~v, was calculated from the total brain radmactlvity minus the terminal plasma concentration of [3H]AIB multiplied by the plasma volume For the determination of the regional plasma volumes, 15 /aCI [14C]dextran (DuPont/NEN. Boston, MA) was injected via the femoral vein 3 mln before the animal was decapitated The plasma volume of the brain (ml/g) is equal to the ratio of the 14C-counts in brain (per g) and plasma (per ml) The [3H]AIB was given 1 rain after completion of the carotid Infusion of 10-6, 10 -s, 2 × 10-s, 5 × 10-s M OlelC acid One group of rats that received 2 × 10 s M olelc acid was Injected with [3H]AIB 80 rain after the carotid infusion to examine the reverslblhty of the blood-brain barrier opening Five rats were used in the control and each experimental group The analysis of variance employing Dunnet's test was used for the statistical comparisons of experimental and control groups
RESULTS T h e p h y s i o l o g i c a l p a r a m e t e r s a r e s u m m a r i z e d In T a b l e I
T h e r e were no differences a m o n g the groups m the
mean
arterial
blood
pressure
(MABP),
arterial
pH,
p C O 2, p O 2 o r h e m a t o c r i t A n i m a l s s u r v i v e d t h e h i g h e r
Fig l Evans blue extravasatlon 15 min after right sided intracaroUd infusion of 2 × 10-5 M oleic acid The appearance of the Evans blue staining is heterogeneous, with pronounced staining In the right cortical and hIppocampal areas and the left parasagmal cortical areas
259
11t
• ACR
+
• PCR +
A
•
e-
HR
E O) ..... -¢
60'
+,
[ ] CER
I--
o a O Ix t,/'}
40
•
ACL
•
PCL
•
HL
20
[] CEL • Bs
_ , _
Control
10-6 M
5x10-5 M
10-5 M
Fig 2 Effect of different concentrations of olelc acmdon BBB permeablhty to AIB Between ] and 11 man following the infusion of 10-s M oleic a o d there was a 10-fold increase m the AIB PS product of the infused hemisphere After 5 x 10-s M oleate, a further increase was observed A C R , PCR, HR, BGR, C E R right sided anterior and posterior cortex, hmppocampus, basal gangha and cerebellum, respectwely ACL, PCL, HL, BGL, CEL similar but on the left smde BS bramstem Data points represent the means + S E M of 5 rats in each group *P < 0 05, **P < 0 01 compared with the controls and 10-6 M oleate +P < 0 05, ++P < 0 01 compared with 10-5 M oleate
The [3H]AIB PS product vaned between 2 and 4 /A/g/mm in the brain regions of the sahne-perfused control rats (Fig 2) These values are stmdar to those reported previously for normal rat brain z2 and indicate that the sudden perfuslon of the rat brain with sahne does not, by itself, cause the BBB to open No change was
observed after infusion of 10 -6 M olelc acid, but a slgmficant, 10-fold increase was measured m the areas of the right hemisphere after the admlmstraUon of 10-s M olelc acid (Fig 2) A further two-fold mcrease m the [3H]AIB PS product in the nght sided brain regions and a significant mcrease m the nght cerebellar hemisphere
40
• ACR • "
PCR
[] HR
30
[]
BGR
[]
CER
0
•
ACL
¢3 O n-
•
PCL
[]
HL
[]
BGL
[]
CEL
E
I'-
(/1
20"
10. +
÷+
o
~ Control
m 2x10-5 M 1-11 mm
BS
2x10-5 M 80-90 mln
Fig 3 Reverslbd~ty of the changes m the blood-brain barrier permeabdlty to AIB Immediately after the infusion of 2 x 10-s M olemc acid there was a large increase m the AIB PS product, however, 80-90 m m following the mfuslon the values returned to the control level Data points represent the means + S E M of 5 rats m each group *P < 0 05 compared with the controls +P < 0 05 compared with 2 x 10-5 M 1-11 mm group There was no slgmficant difference between the control and the 2 x 10-s M 80-90 m m groups
260 TABLE I Phystologtcal parameters
Values are means ± S E M of 5 animals
MABP (mm Hg) Temp (°C) ArterlalpH p~CO2 (mm Hg) PaO2 (mm Hg) Hematocrlt (%)
Control~
10 6M
10 5 M
2xlO
97 ± 6 365+01 7 37 + 0 01 45 ± 1 77 ± 3 36 ± 1
87 ± 5 367±02 7 35 + 0 01 46 + 4 73 ± 3 37 ± 1
90 ± 4 365+01 7 35 ± 0 02 38 + 5 72 ± 7 33 _+2
87 ± 5 367±01 7 36± 0 02 43 ± 3 84 ± 5 36 ± 2
and the left corttcal areas was observed after Infusion of
SM
5×10 SM
2xlO~M+ 80 m m
91 ± 5 365±01 7 31 ± 0 02 41 ± 7 72 ± 5 34 _+4
92 _+6 36 8 ± 0 ! 7 34±0 01 46 _+2 80 ± 5 36 ± 2
DISCUSSION
5 × 10-5 M olelc acid (Fig 2) The reversibility of the oleic acid-induced BBB openlng to [3H]AIB was examined 1-11 and 80-90 mln following the mfuston of 2 × 10-5 M oletc actd Thts dose caused an lntttal Increase In the A I B permeablhty of the same magnitude as a dose of 10-5 M, however, by 80-90 m m later, the permeabthty returned to the control values (Fig 3) A higher [a4C]dextran space was measured in the right stded cerebral regtons parallel wtth the mcreased A I B permeablhty, an Increase that was statistically stgnificant in the animals perfused with 5 x 10-5 M olelc acid (Table |I) This increase most likely represents extravasatton of
Olelc acid increases BBB permeabthty m a dosed e p e n d e n t , reversible m a n n e r The mcrease m the A I B PS product in the cortical regions of the left hemisphere and right cerebellar hemisphere can be explained as a result of cross perfuston to the contralateral hemisphere and tpsllateral cerebellar areas as lndtcated by the pattern of the extravasated Evans blue The heterogeneous Evans blue staining indicates an u n e v e n distribution of areas with Increased a l b u m i n p e r m e a b l h t y As the flow rate of the Infusion was high, no streaming p h e n o m e n a can be expected 28, and the cause of the u n e v e n appearance of the a l b u m m staining remains to be explamed
[a4c]dextran as a result of the extenstve BBB opening rather than a true increase In plasma volume Conse-
Smce the BBB is created by the plasma m e m b r a n e and ttght junctions of the endothelial cells, the change m BBB
quently, the values of A I B permeabthty at the higher
permeabthty caused by olelc acid might be explained as
doses of olelc a o d are probably underestimates of the true permeablhty
a h p l d - m e m b r a n e mteractlon The experiments of Raz and Llvne 26 d e m o n s t r a t e d that the osmotic fraglhty of erythrocytes is affected by olelc a o d Low concentrations
TABLE II Effect o f oletc acM on [14C]dextran space
Values are means ± S E M of 5 animals and expressed m units of/~l/g ACR, PCR, HR, BGR, CER right sided anterior and posterior cortex, htppocampus, basal gangha and cerebellum, respectJvely ACL, PCL, HL, BGL, CEL slmdar but on the left slde BS bramstem
ACR PCR HR BGR CER ACL PCL HL BGL CEL BS
Contro~
10 ~ M
IO- S M
2 x lO-S M
5 x 105M
2 × 10 ~ M + 80-~ mm
69±05 70±05 70±08 73±09 124±14 73±05 78±07 87±06 71±04 118±08 94±09
69±05 70±05 82±10 80±04 121±21 75±02 87±06 81±10 84±07 132±12 90±06
130±20 142±21 132±13 123±12 131±11 83±09 94±11 94±13 89±04 125±16 111±04
119±11 147±07 146±13 126±03 134±13 90±06 92±06 91±15 83±06 124±09 148±40
272±76* 307±77* 352±101" 279±70* 173±26"* 173±56"* 133±45 104±30 99±25 134±04 97±07
83±14 74±09 76±06 76±03 98±06 66±02 85±07 68±04 77±05 107±06 89±04
*P < 0 01 compared with the controls and the 10-s M group **P < 0 05 compared with the controls and the 10 s M group
261 reduced the proportion of cells that were lysed while higher olelc acid concentrations (10-5 M and higher) increased their susceptlblhty to osmotic shock In our experiments the same threshold concentration range of olelc acid was found to be damaging to the BBB Olelc acid IS an amphlphlhc substance which can induce major changes In membrane function 1° At low concentrations, as a monomer, ~t inserts itself into the lipid membrane, changing the physical properties and physiological function of the membrane m a way that appears as a membrane-stablhzmg effect ~° At high concentrations, however, amphlphdlc monomers aggregate into mlcelles which have the ablhty to incorporate membrane hplds into their structure The incorporation of high concentrations of amphlphlles into membranes physically &srupts the hpld bllayer and, consequently, destroys the integrity of biological membranes 1° If an olelc acidmembrane interaction is responsible for the increased permeability of the BBB, It is surprising that the process is so rapidly reversible Olelc acid is also known to be a potent actwator of protein klnase C 13'14, a key enzyme in mediating transmembrane signahng 16 The presence of protein klnases in the brain capillaries has been shown previously22 and modulation of the kinetics of protein phosphorylatlon by second messengers may contribute to the regulation of the BBB permeablhty 2~ Although there is only indirect ewdence that protein klnase C may play a role m the regulation of the BBB transport processes 9, the possibility exists that ole~c acid may affect the permeablhty of the brain vascular endothelial cells by changing protein klnase C-induced protein phosphorylatlon The rapid reverslbdlty of the olelc acid-reduced BBB opening supports the view that a reversible biochemical process ~s the underlying event Recent stu&es 1~ 18 20 have revealed yet other biological effects that are exerted by olelc acid that could influence the permeabdlty of the BBB Reduced synaptosomal Na+,K+-ATPase actlwty 2° and inhibition of the mltochondrml oxidative phosphorylatlon 19 were observed after treatment with ole~c acid The release of reactwe oxygen metabohtes may also be revolved m barrier
destruction as olelc acid-induced pulmonary edema can be attenuated by pretreatment with catalase and superoxide dlsmutase 11 Olelc a o d in cortical superfusion experiments performed by Unterberg et al 29 was found to be without effect on the BBB of pml vessels Given lntracerebrally it did not induce changes in brain water and cation levels 3 The discrepancy between our results and those of others may be explained by the different route of administration of the olelc acid Increased olelc acid concentrations were measured in the plasma and interstmally drained brain edema fluid in cats exposed to cold InJury I and in the brain tissue following lschemla s'~5 30 Our results Indicate that the elevated olelc acid concentration might contribute to the &sturbance of BBB permeablhty observed in these experimental models However, binding of olelc acid to albumin would normally reduce the concentration of free olelc acid m the plasma by a considerable amount In our studies, the lntracarotld infusion rate was high enough to completely clear the blood from the carotid circulation on the side of the infusion Thus, there was no binding of olelc acid to albumin during the infusion and the BBB was exposed to the stated concentrations of free olelc acid We are unable to predict whether albumin-bound olelc acid would have a similar effect In conclusion, mtracarotld infusion of olelc acid increases BBB permeablhty m a dose-dependent and reversible fashion The procedure is well tolerated in the short term, however, neither long-term toxicity nor localized effects leading to cellular injury, brain edema, and metabolic disturbances have been examined It is unclear at the present time whether the BBB opening reduced by oleic acid results from a non-specific lipidmembrane interaction or the actwat~on of a specific biochemical pathway Further studies are warranted since this procedure may provide a useful clinical approach to modification of BBB permeablhty
Acknowledgements The authors thank Gloria Rodnguez for her valuable secretarial support This work was supported by Grant NS 23870 from the National Institutes of Health
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