Opening the blood-brain barrier to zinc

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Opening the blood-brain barrier to zinc J R Blair-West, D A Denton, A.P. Gibson and M.J McKinley Howard Florev Institute ol Erpenmental Physiology and Medicine, Umverslty of Melbourne, Parkvdle, Vie ( 4ustraha) (Accepted 13 June 1989)

Key words Cranial blood osmolahty Electroconvulslve shock, Blood-brain barrier, Sheep Zinc, Cerebrospmal fluid

Sheep with guide tubes implanted over the brain lateral ventricles, in order to facilitate episodic sampling of cerebrospmal fluid (CSF), were used to determine the effects of increasing cramal blood osmolallty or electroconvulslve shock (ECS) on the permeability of the blood-brain burner (BBB) to zinc Zinc acetate solutmn (1 mg Zn/ml) was infused intravenously (l v ) at I 0 ml/mm for 30 mm and then continuously at 0 125 ml/mm This infusion increased plasma total zinc concentration (pZn) approximately 10-fold without altenng CSF zinc concentration (CSFZn) After 1 5-3 5 h, 4 M NaCl was refused at 5-10 ml/mln for 10 mm into one carotid artery with the other carotid artery occluded, or the animals were anaesthetized and given an ECS (140 V, 2 s) Paired samples of blood and CSF were collected before and after these treatments Results were 0) CSFZn was approximately one tenth of pZn, (n) zinc administered l v was almost completely excluded from the CSF, (Ul) increased cranial blood osmolahty or ECS increased CSFZn in all experiments, but the time course and extent of the rise were variable CSFZn reached the concentrations of zinc in normal sheep plasma in some experiments, (iv) CSFZn subsequently fell towards the low values of zinc in normal CSF, (v) the animals suffered no evident ill-effects from either procedure The procedures may, therefore, be used for reversible opening of the BBB to particles such as zinc in conscious or anaesthetized sheep with no troublesome sequelae into the brain, as m e a s u r e d m c e r e b r o s p m a l fluid (CSF),

INTRODUCTION

d u n n g lntracarotld infusion of hypertonic N a C I solution and I n c r e a s e d cranial b l o o d o s m o l a h t y z4-zT, e l e c t r o c o n v u l -

d u n n g E C S T h e purpose in selecting zanc as a m a r k e r of

s i r e s h o c k ( E C S ) (for r e v i e w s see refs 9, 10) and o t h e r

B B B permeability was directed to its role m m a n y metal-

m a n o e u v r e s i n c r e a s e the p e r m e a b l h t y o f t h e b l o o d - b r a i n

l o e n z y m e s c o n c e r n e d with D N A rephcat~on, r e p m r and transcnptton 6 7 16 32 (for reviews see refs 2, 11, 19, 23, 28,

b a r r i e r ( B B B ) 8 T h e t e c h n i q u e u s e d m o s t f r e q u e n t l y in e x p e r i m e n t a l a m m a l s has b e e n the infusion of c o n c e n -

30, 31), its possible role in neurotransmlsslon ~429 (for

t r a t e d s o l u t i o n s of n o n - e l e c t r o l y t e s , e g ,

review see ref

mannltol or

a r a b l n o s e , into cranial a r t e r i e s In a n a e s t h e t i z e d p r e p a r a tions T h e m e c h a n i s m o f o p e n i n g the B B B in this way has

23) and behavaoural disorders 12x3 (for

reviews see refs 2, 30), and o u r interest in specific trace e l e m e n t deficiencies in ruminants 1'2°

b e e n h y p o t h e s i z e d as c e r e b r o v a s c u l a r dilation, s h r i n k a g e o f t h e v a s c u l a r e n d o t h e h u m , and o p e n i n g the 'tight j u n c t i o n s ' b e t w e e n e n d o t h e l i a l cells e5 r a t h e r than plnocytosls

O s m o t i c o p e n i n g o f the B B B has b e e n d e m -

o n s t r a t e d w~th m a r k e r s such as E v a n ' s blue o r p r o t e i n s but s o m e studies using drugs, e g m e t h o t r e x a t e 22, inc l u d i n g h u m a n s u b j e c t s zI, h a v e i n d i c a t e d large increases m t h e c o n c e n t r a t i o n of this a g e n t in b r a i n tissue P r o c e d u r e s for r e v e r s i b l e o p e n i n g o f the B B B m a y p r o v e useful for the d e l i v e r y of t h e r a p e u t i c agents into the brain T h e r e are o t h e r potential uses, e g , the delivery Into the brain of specific nutrients, e g , trace elements, or agents that m a y modify appetitive behaviours such as hunger. N a appetite and thirst 5 T h e r e f o r e the capability o f reversibly o p e n i n g the B B B by hyperosmotIc and E C S techniques has b e e n investigated in sheep First attention has b e e n given to the penetration of zinc

MATERIALS AND METHODS Seven adult cross-bred female Merino sheep, weigbang 30-42 kg, were used The animals were housed in individual metabolism cages and fed 0 8 kg/day of a mixture of alfalfa and wheat chaffs They had free access to water and food except dunng experiments Each animal had bilateral carotid artery loops, i e . the carotid arteries in the mid-neck region were cleared of any branches and enclosed in a tube of skin, made from folds in the neck The arteries were then available for infusion and restriction of the cranial blood flow Each sheep had a guide tube (17 gauge stmnless-steel Luer Lock needle, shaft length 21 mm) implanted approximately 6 mm above each lateral bram ventricle This surgical preparation has been described ~7 Stainless-steel probes (0 8 mm bore) could be introduced through these guide tubes into the ventricles for the purpose of CSF sampling in conscious animals standing in their cages On the day of experiment, a blocker was removed from one of the guide tubes and a probe of appropriate length was introduced into the lateral ventricle The probe was connected to a polythene cannula and a CSF sample of approximately 1 ml was withdrawn under gravity The probe was left in place throughout the experiment and

Correspondence J R Blair-West, Howard Florey Institute of Experimental Physiology and Medicine University of Melbourne, Parkvllle Vlc 3052, Australia

0006-8993/90/$03 50 © 1990 Elsevier Science Publishers B V (Biomedical Division)

RESULTS

CSF samples were collected as required On the day before the experiment, polyethylene cannulae w e r e placed m both jugular veins, one for infusion of zinc acetate solutton and one for blood sampling The cannulae were filled with dilute heparln solution On the experimental day one or two control samples of blood and CSF were taken and then zinc acetate solutmon m 0 9% saline (1 mg zlnc/ml) was infused intravenously at 1 ml/mm for 30 mln and then at 0 125 ml/mln for the rest of the experiment The loading mtus~on increased plasma total zinc concentration from 0 5-1 mg/I to 7-10 mg/I and the slower rate of mfuston maintained this level for the remainder of the experiment Two to 4 paired samples of blood and CSF were collected during 1 5-3 5 h of infusion Then an infusion lane was introduced into the left carotid artery and the right carotid arter'y was occluded Twenty min later, the left carotid artery was constricted until palpation md~cated that the blood flow "~as r e d u c e d and a solution of 4 M NaCI was refused into the left carotid artery at 5-10 ml/mm for 10 min The obstructmons on the carotid arteries were then removed Paired samples of blood and CSF were collected for up to 4 h after the NaCl infusion In some experiments it was possible to collect only 2 or 3 CSF samples during this period (see Results) In 4 experiments the zinc acetate infusion procedure was the same but, instead of the NaCI infusion, the animals were taken to the operating theatre, anaesthetized with 1 v thlopentone then halothane/oxygen and, after 0 25 mg/kg I v scoline they were given an electroconvulsive shock (140 V, 2 s duration) with electrodes placed on the temples Breathing was regulated by a Campbell venhlator at 8-10 breaths/mmn during these procedures The anaesthetic was stopped and the animals were awake and standing in their cages 1 h later The zinc acetate infusion was interrupted for 2 periods ol 30 s, going to and from the theatre Zinc concentrations in plasma and CSF were determined by atomic absorption spectophotometrv (Perkin Elmer 272 for early experiments and then Vanan SpectrAA-40) The method for plasma was a modification of the method of Butrimovitz and Purdy" and for CSF a modification of the method of Meret and Henkm Is was used Zinc acetate solutions for infusion were prepared m 0 9,% sahne Results are quoted as mean +_ S E M and statistical comparisons w e r e performed by 'paired' t-test

Increased crantal blood osmolahtv Two experiments measured

representing

(CSFZn)

are shown

6-

(.and-)

4-

1

In o n e

p l a s m a zinc c o n c e n t r a t i o n ( p Z n ) f r o m 0 8 t o 10 0 mg/l a n d t h e s l o w e r r a t e o f anfuston t h e n m a i n t a i n e d p Z n at 8 9 - 9 7 rag/1 t h r o u g h o u t t h e e x p e r i m e n t , i e , u n t i l C S F s a m p l e s c o u l d n o t b e c o l l e c t e d d u e to s o m e o b s t r u c t i o n at t h e t~p o f t h e p r o b e

C S F Z n w a s s t e a d y at 0 0 5 - 0 (17

mg/l u n t i l t h e c o m p l e t i o n o f t h e 4 M N a C I l n t u s l o n , a n d t h e n rose to 0 67 mg/l in 15 m m

C S F Z n h a d f a l l e n to 0 20

mg/l 1 h l a t e r In t h e o t h e r e x p e r i m e n t , p Z n r o s e f r o m 0 7 t o 8 9 mg/I d u r i n g t h e l o a d i n g i n f u s i o n ot zinc a c e t a t e a n d v a r i e d f r o m 6 7 to 9 5 mg/l f o r t h e r e s t o f t h e e x p e r i m e n t C o n t r o l C S F Z n w a s 0 15 mg/l a n d w a s a g a i n u n a l t e r e d by zinc a c e t a t e i n f u s i o n the 4 M NaCI

CSFZn

increased gradually alter

i n f u s i o n to a m a x i m u m

ot 0 40 mg/1

approximately 1 h later and was higher than control for another 3 h In 7 e x p e r i m e n t s (Fig

2) m e a n c o n t r o l p Z n w a s () 64

+ 0 05 mg/l a n d m e a n C S F Z n w a s 0 053 + 0 021 mg/l In samples taken 5-20 mm before the infusion ot 4 M NaCI, mean

p Z n was 8 8 _+ 0 3 mg/l a n d C S F Z n

+ 0 020 mg/l (n s )

CSFZn

was 0 070

responses after 4 M NaCI

infusions were variable m time course and degree

not

solely d u e t o d i f f e r e n c e s m s a m p h n g t i m e o r p r o b l e m s

10-

Plasma Z,nc Conc

an Fig

e x p e r i m e n t , t h e l o a d i n g i n f u s i o n o f zinc a c e t a t e i n c r e a s e d

CSFZn

f r o m b a s e l i n e in all e x p e r i m e n t s

8-

ot t h e

e f f e c t s o f 4 M N a C I i n f u s i o n o n C S F zinc

concentration

with obtaining CSF samples

mg/I

the extremes

rose substantially

The delay varied from

-1 0

-0 8

mg/I

-0 6

CSF Zinc Conc

- 0 4 (oand~)

b-- ;-----:-:o"J

2-

Infus,on of 4M NaCI ,nto constricted L carotid artery R carotid artery occluded

-~ lmg/m,n IV~Zn~ -

-

-

1

-

-02 o

Infusmon T

2

0 125mg/ml mmn L

V

]

T

3

4

5

r

6

7

Hours Fig 1 Two experiments (closed circles and open circles, hlled triangles and open mangles) Changes in the concentration of zinc in plasma (filled symbols) and cerebrospmal fluid (CSF) (open symbols) after (1) intravenous (i v ) infusion of zinc acetate solution and then (n) occlusion of the right carond artery, followed 20 mln later by (nl) constriction of the left carotid artery and infusion of 4 M NaCI into the left carotid artery at 5-10 ml/min

12

The response was delayed tor 35 mm or less m experiments and for more than 1 h nl one experiment Maxamal C S F Z n values were reached in 35 to approximately 100 mln C S F Z n values 3 5 h after ECS were stdl hagher than control in 3 experiments Fhe mean p Z n at that time was 10 3 -+ 0 6 mg/l

10

mg/I

0 8-

CSF Zinc Conc

0 6 O4 02

•

O-

*

~

DISCUSSION

• I

to

constricted L c a r o t i d artery R carotid artery occluded

Hours

Fxg 2 Seven experiments The concentration of zinc m cerebrospinal fired (CSF) m control samples (crosses) and samples during intravenous infusion of zinc acetate solution, before (closed mrcles) and after (open circles) mfusmn of 4 M NaCI into the left carotid artery w]th right carotid artery occluded and left carotid artery constricted 5 to 30 m m after completing the 4 M NaCl infusion M e a s u r e d p e a k values were reached 5-65 min later Values d e c h n e d slowly or rapidly after the p e a k The last C S F Z n value r e c o r d e d m each e x p e r i m e n t , 1-4 h after 4 M NaCI Infusion, were still above baseline in 6 of 7 e x p e r t m e n t s The m e a n p Z n was 9 6 _+ 0 7 mg/l at that nine

Electroconvulstve shock In 4 e x p e r i m e n t s , m e a n control p Z n was 0 73 + 0 06 mg/1 Zinc acetate infusion had mcreased p Z n to 8 8 + 0 5 mg/l m blood samples taken 30 mm before the E C S M e a n control C S F Z n was 0 090 + 0 038 mg/l and after zinc acetate mfusmn, but shortly before ECS, it was 0 068 + 0 027 rag/1 (n s ) (Fig 3) All experiments were c o n t i n u e d for 3 5 - 5 h after ECS because there were no difficulties with C S F sampling C S F Z n rose m all experiments after E C S , with varmble time courses and m a x i m a

1210-

mg/I

0 8-

CSF Zknc

06-

Conc

0402-

I

2

OECS 1 4 0 ' 4 , 2 sec

-4

-'2

0

Ftg 3 Four experiments The concentrauon of zinc in cerebrospmal fired (CSF) m control samples (crosses) and samples durmg intravenous lnfusmn of zinc acetate solution, before (closed circles) and after (open orcles) electroconvulswe shock (140 V 2 s)

CSF zinc concentration in sheep was a p p r o x i m a t e l y one tenth of the total concentration of zinc m plasma This p r o p o r t i o n Is m accordance with the values m man t8 C S F Z n was u n a l t e r e d by increasing p Z n a p p r o x i m a t e l y 10-fold by the intravenous infusion of zinc acetate solution It as not known how much of this a d d m o n a l p Z n was bound or free, but most of the zinc in normal plasma is bound to protein 3° 31 with about one third firmly bound to a2-macroglobuhn Thus. it is not possible to conclude that the exclusmn of a d m i n i s t e r e d zinc from the C S F was solely due to a high reflectance of zinc in the chorold plexus or brain capillary e n d o t h e h u m The exclusion of administered zinc from the C S F a p p e a r e d to be almost complete and this result is consistent with the slow p e n e t r a n o n of an antrapentoneal pulse of 65Zn into rat brain and C S F 15 Despite this evidence for low p e r m e abdaty of the BBB, zinc levels in h u m a n and rat brain are second only to iron amongst the trace elements 23 Zinc deficiency did not reduce the brain total concentratmn of zinc 23, zinc retention m a y in fact increase is Increasing cranml b l o o d o s m o l a h t y by mfusmn of 4 M NaCI mto a carotid artery increased C S F Z n 2 - a p p r o x i mately 40 fold with varmble time courses of rise and fall In 3 experiments, the C S F Z n reached the normal range for p Z n A reduction of c a r o n d b l o o d flow was necessary for this response because 2 p r e h m m a r y experiments without the reductmn showed no effect of l n t r a c a r o n d 4 M NaC1 mfusmn on C S F Z n A s s u m i n g that the carond blood flow was r e d u c e d trom 500 to 250 ml/mm, the infusion of 4 M NaCI at 5 - 1 0 ml/mm would have increased the plasma [Na] m the blood to the head by a p p r o x i m a t e l y 100-200 mmol/i The change may have been less than this because the collateral and vertebral blood supply to the brain in these circumstances must be substantial, as indicated by the observation that at is possible to occlude one carotid artery and then gradually occlude the o t h e r without dmtressmg most sheep (unpubhshed observations) The BBB of the rat has been shown to open above a threshold o s m o l a h t y of 385 m O s m o l / k g 4 This value appears certain to have been e x c e e d e d in the present experiments when they included reduction of carotid blood flow The 4 M NaCI infusion being c o m b i n e d with &tarnished carotid blood supply, at as possible that hypoxia may

have contributed to the effect of the infusion on C S F Z n

osmotic shrmkage of the space or ot the brain itsell

H o w e v e r , constrlcuon of the contralateral carotid artery

Taking these matters of variations in sampling time,

for 20 m m before the infusion of 4 M NaCI did not alter

strength ot stimulus, and site ot CSF sampling into

CSFZn

account, it ts still likely that some of the varlabthty in

and

the

carotid

obstructions

were

removed

i m m e d i a t e l y after the infusion T h e extensive vertebral

responses to treatments was due to indwidual sheep

and collateral blood supply to the brain may also have

variation

a m e l i o r a t e d any ettects of local hypoxaem~a

this was not tested

T h e responses of C S F Z n to E C S were m many ways

In view of the n u m b e r ol variables revolved H o w e v e r , the results show clearly

that both treatments caused the entry of zmc from the

In two

plasma mto the C S F "[ he entry of zinc was not down the

e x p e r i m e n t s the responses were rap~d and in one it was

track of the ventrlcular probe because 10-fold increase m

delayed for an hour T h e maxima of C S F Z n were in the

p Zn did not alter C S F Z n m any e x p e r i m e n t before

range of normal p Z n in 3 experiments In 2 experiments

t r e a t m e n t with 4 M NaCI or ECS

the effect was transient and m 2 experiments tt was

or almost to, baseline values after treatments despite

p r o l o n g e d for 2 -3 h

continuing high p Zn T h e r e f o r e the t r e a t m e n t s must have

similar to the responses to 4 M NaC1 infusion

The variability of C S F Z n responses to treatments may

Also, C S F Z n fell to,

o p e n e d the BBB to zinc and the opening was reversible

have several explanatmns With 4 M NaCI infusion CSF

as indicated by the decline o | C S F Z n during the post

samples were not or could not be obtained at exactly the

t r eat m en t period of observation

same ttmes but there were sufficient samples taken in the

treatments also o p e n e d the B B B to protein and that some

first 30 rain after infusion to show that C S F Z n rose

of the zinc appearing in CSF e n t e r e d with this protein

rapidly in some ex p e r im e n t s and little, or not at all, in

It is possible that the

Th e reducUon of C S F Z n after the peak value, as well

others The same situation apphed to E C S experiments

as indicating reduced rate ol entry as the BBB closed,

T h e r e was only a brief rise in C S F Z n in some 4 M NaCI

suggests clearance ot zinc by uptake into brain tissue,

and E C S experiments, and in other experiments the rise was sustained for hours T h e r e was some variation m the

possibly transport out of the CS F c o m p a r t m e n t by the chorold plexus is and dilution by ongoing secretion ot

rate of infusion ot 4 M NaC1, ranging trom 5 to I0 ml/mln

CSF

through the 10 rain of infusion and carotid blood flow was always reduced but probably not equally so T h e ECS

Th e findings provide new information on two procedures that may be used to open the BBB reversibly for

h o w e v e r had constant p a r a m e t e r s A n o t h e r variable was

experimental

the location of the samphng probe m the lateral ventricle

suffered no post-experimental physiological damage or

A l t h o u g h the guide tubes are positioned stereotaxlcally ~7

changes of d e m e a n o u r that could be detected by obser-

there must have been some variation in the position

vation or m e a s u r e m e n t of daily tood and water retake

purposes

The

sheep

that

were

used,

above the lateral ventricles and also in the depth of the

Further experiments are required to d em o n st r a t e the

sampling probe in the ventricle Samphng from different

quantttattve relations b et w een the strength and duration

sites m different sheep may have contributed to the

of stlmulaUon and the extent of BBB opening been d e t e r m i n e d by others 26

varlabthty ot C S F Z n after treatments

"Ihe inability to

as have

collect CSF samples after 4 M NaCI infusion in some e xpe ri m en t s may have been caused by reduction oI the ventrlcular space around the tip of the probe due to

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A{l~no~ledgement This work ,has supported by a grant from the Natmnal Health and Medical Research (ouncd of Austraha

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