A minimally invasive technique for multiple measurement of regional blood flow of the rat brain using radiolabeled microspheres

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A minimally invasive technique for multiple measurement of regional blood flow of the rat brain using radiolabeled microspheres Masatsugu Nakal 1, Kmya Tamakl 1, Jm Yamamoto 1, Aklto Shlmouchl 1 and Masanobu Maeda 2 INattonal Cardtovascular Center Research Insntute, Sutta, Osaka (Japan) and 2Department of Phystologv, Osaka Ctty Umverstty Medwal School, Abeno, Osaka (Japan)

(Accepted 26 September 1989) Key words Cerebral blood flow, Mlcrosphere method, Brachml artery, Cross circulation, Hypercapma

To observe the rat brain blood flow with mlcrospheres, a left venmcular catheter for sphere introduction was threaded into posmon wa the right brachml artery Cross-orculauon with a donor rat was employed to prevent any mismatch between the refused and sampled blood volumes Muttlple measurements yielded stable flow data and the responsweness to hypercapma was well preserved, lrrespectwe of the dose of rejected spheres (48 × 103 and 130 × 103) Whtle the mlcrosphere method has been extenstvely employed m larger antmals for bram blood flow measurement, use of thts techntque m rats is less popular The major drawbacks inherent m the method apphed to rats may be the lnvaslve surgery required for cathetertzat~on for mlcrosphere injection, the burdensome attention necessary for mamtenance of the hemodynamlc state, and the paucity of knowledge of an adequate n u m b e r of spheres to be rejected In the present study, we attempted to develop a mmtmally mvaslve m~crosphere method suitable for measuring the regtonal blood flow of the rat brain Experiments were conducted on 24 male Wtstar rats weighing 248 + 30 g (mean + S D ) Under 2% halothane anesthesm, two tapered polyethylene catheters (0 96 m m m ortginal outer dtameter) were threaded into the femoral artertes btlaterally A short segment (7 cm) of catheter (0 61 mm m outer dmmeter) was telescoped into the parent catheter of 0 96 mm in outer dmmeter After drawing back the lateral edge of the right pectoral muscle medtally, the catheter was threaded into the rtght brachtal artery so that ~ts ttp was located m the left ventrtcle The animals were then anesthetized with chloralose (12 5 mg/kg, l p ), venttlated wtth a gas mtxture of 0 2 and N 2, immobilized with d-tubocurarme (0 5 mg/kg, 1 m ), and kept m a homeothermtc state at 37 °C No further surgtcal mampulatlons that might be noxious to the antmal were performed We next set up a cross-ctrculatton between the rat under study (the reoplent) and a consoous donor rat The d o n o r was chromcally provided with arterml and

venous catheters threaded mto the thoractc aorta and left superior vena cava, respectively H e p a n n of 600 U/kg t v was administered to both rats A mmlmal requtrement of heparm m the present study may be at thts dose, smce coagulation of blood would otherwtse begm within an hour wlthm tubings employed for the cross-ctrculatton The cross-clrculatton allowed blood from the rectplent's femoral arterial catheter to dram into a samphng tube Fresh blood from the donor's arterial catheter was allowed to drain mto the rectplent's ventrtcular catheter, through whtch spheres were tnjected The blood flow in both dlrecttons was mamtalned at the same rate (1 2 ml/mm) by employing a double channel tube pump (Mmtpulse 3, Gllson, France) To overcome the blood loss by the donor rat during such cross-ctrculation, blood previously sampled from an outbred rat was introduced manually into the donor wtth a syringe through the venous catheter The mlcrospheres (15 +- 1 5 / x m m diameter, NEN) were labeled with la3Sn, 14lEe, 8SSr, or 465c, and suspended m 0 9% sahne with 0 01% Tween 80 A known number of spheres was kept in a segment of stlasttc tubing of 50 /~l in lummat volume 4 After sontcation, the tube segment was interposed between the rectptent's ventrtcular catheter and the catheter leading from the donor's aorta The cross-circulation was then allowed to flow for a pertod of 60 s During the first 10 s, we patted the tube segment w i t h a metal spatula to ensure excellent mtxmg of the spheres wtth the flowmg blood Begmnmg stmultaneously with the start of the lnjectton of spheres, a reference blood sample was

Correspondence M Nakal, Nahonal Cardiovascular Center Research Institute 5-7-1 Fujlshlro-dal, Smta, Osaka 565, Japan

0006-8993/90/$03 50 ~ 1990 Elsevwr Science Pubhshers B V (Biomedical Dwlslon)

169

n u m b e r of spheres was injected, the r e p r o d u c l b i h t y of 4 successive m e a s u r e m e n t s of b l o o d flow u n d e r n o r m o c a p nla (/),,CO 2 = 36 7 mm H g on average) was excellent (test for overall differences over time 1~, Ftg 1, u p p e r section) Stattstlcally there was no difference m the responstveness of the blood flow to h y p e r c a p n l a (p ,CO: = 62 0 m m Hg on average, n = 20) b e t w e e n those o b s e r v e d at the 2nd and 4th injections ( P > 0 05, patred t-test) in each brain region (Ftg 1 lower section) No signlhcantly large difference was noted within any pmr of flow data o b t a i n e d at the smaller dose and larger dose ot mjected spheres (P > 0 05, u n p a i r e d t-test) Mean artertal blood pressure, and gas tenstons and p H ot arterial blood during individual e x p e r i m e n t a l settmgs were hsted m Table I Mlxmg of the spheres with b l o o d was round to be a d e q u a t e m every case smce b l o o d flow was symmetrically dtstrlbuted to the ktdneys In the mlerosphere m e t h o d p r o p o s e d previously for brain blood flow m e a s u r e m e n t s m rats, extensive thoracotomy or obstructton of the rtght c o m m o n carotid artery was necessary for p l a c e m e n t of the c a t h e t e r in the left atrium ~ or left ventricle 2 o ~ m for the lnjectton ot spheres A c u t e unilateral obstruction ot the artery has been r e p o r t e d to blunt the responstveness ol the brain blood flow to h y p e r c a p n t a severely 2 In the present study, it was found that acute p l a c e m e n t ol the mjectton catheter m

p u m p e d from the rec]plent's femoral arterial catheter into a plastic tube The cross-circulation was also emp l o y e d for frequent b l o o d sampling to m o m t o r the blood gas values and p H M l c r o s p h e r e s were injected 4 times to examine the r e p r o d u c l b l h t y of the regional b l o o d flow m e a s u r e m e n t s tn the n o r m o c a p n l c state (14 rats), and the reproducibility ot observations of the responsiveness of the flow to h y p e r c a p n l a induced by 5% C O 2 inhalation (10 rats) F u r t h e r , each of these two groups of rats was subjected to e x p e r i m e n t s involving injections of a smaller or larger dose of spheres The animals were then sacrificed b} h e m o r r h a g e The cerebral cortex (620 mg on average n = 24), lower bralnstem (207 mg, pons and medulla) and h y p o t h a l a m u s (52 mg) were dissected out bilaterally from the b r a m Kidneys were also excised The tissue blood flow and the numbers of spheres lodged in each Ussue were then calculated by a standard m e t h o d (e g , rels 4, 6) In the e x p e r i m e n t s under n o r m o c a p n i a with a smaller dose of mlcrospheres (48 x 10 ~ beads on average), the n u m b e r s of spheres t r a p p e d in the cerebral cortex at each mjectlon were 500 beads on average, in the lower b r a m s t e m , 207, and m the hypothalamus, 26 Wtth a larger dose (130 x 103 beads), the numbers were 1590, 579, and 69 beads respectively We round that whichever

NORMOCAPNIA Cerebral

cortex

bramstem

Lower

Hypothalamus ....

Large dose ( n = 7 )

100

8

5o

,g 0

E

NORMOCAPNIA (1st & 3rd) & HYPERCAPNIA (2nd & 4th) 0

400

"

•

• *

,

* ,

"0 0 0 J:3

I

Is,,,,,,, do,,, ~o=s~

ILarge

dose I n = 5 )

¢

f0 O~

200

-

n-

0 1

2

3

4

1

Injection

2

3

4

1

2

3

4

number

Fig 1 Regional blood flow (mean --+ S D ) resulting from 4 successive injectlom, (S mln apart) of tile sma[] dose (48 × 10~) ot mmrospheres or the large dose (130 × 10~) of mlcrospheres (10 rain apart) m rats Significant ddfcrcnce~ (*) were lound onb between the flow data obtained under normocapma and under hvpercapma (P < 0 ()()1, paired t-te~t)

170 FABLE I Mean artertal blood pressure, and gas ten ~ton5 and p H oJ arterial blood daring determmanon o f brain blood/lon

Values are means _+S D MAP, mean arterial blood pressure, S, the experiment with a small dose of mmrospheres L with a large dose n = 7 m the normocapmc experiment w~theach dose and 5 m the hvpercapmc experiment w~theach dose Experimental run

MAP(mm Hg) pH~ p~CO2(mmHg) P.O2 (mm Hg)

Normoeapnta

S k S L S L S L

Hvpercapnta

1st

2nd

?rd

4th

123+6 119_+7 738+-002 738+-004 372+38 361+13 135+-14 146 _+ 17

121+6 120+-8 736+002 739+-004 370+1 4 356_+08 132_+13 144 +- 13

114+5 122+-10 737_+001 738+004 375_+29 363_+20 134_+13 142 + 14

121+4 120-+5 737+002 738_+003 374+1 6 363+14 133_+17 140 _+ 15

S L S+ L~ S+ L~ S L

1st

2nd

~rd

4th

113+8 109_+14 737+-003 738+003 38 1_+1 5 368_+12 131_+30 143 _+26

108+_13 114+-4 720+002* 720+003* 615+17" 624_+12" 140_+24 141 _+32

110+7 112+_13 738_+002 737-+003 354_+1 4 z;g9_+06 144_+20 151 +- 33

111+10 118+-5 719_+001" 719+-002" 61 8 + 2 3 " 621+10" 151+_22 142 _+30

Slgmflcant variation across Ume (P < 0 001 test tot overall &flerences over Ume) * Slgmflcantlydifferent from a value during normocapma (P < 0 001 pmred t-test)

the left ventricle via the right brachml artery could be easily achieved and was minimally mvaswe The responsiveness of the blood flow to hypercapma was found to be well preserved with our technique If the v e n t n c u l a r and femoral arterial catheters are exteriorized through a s u b c u t a n e o u s t u n n e l at the nape of the neck, experiments m conscious u n r e s t r a m e d rats wdl be possible Different from the c o m m o n l y utilized method 4 ~ 9 we adopted a cross-circulation for the rejection and rlnsmg out of spheres with fresh blood drained from a d o n o r rat, and also for simultaneous reference blood sampling The blood employed for m o m t o r m g of the gas values was also sampled frequently using th~s cross-circulation The blood flow m both directmns was maintained at the same rate O u r techmque therefore prevented any mmmatch between the blood volumes draining into and sampled from the rat, and was considered especially statable for making repeated m e a s u r e m e n t s of the brain blood flow of such small ammals hke rats We consider that, for rinsing out of spheres by the cross-c~rculatmn, it may not be desirable to use prevmusly sampled autologous blood or pooled blood collected previously from another rat This is because inevitable hypovolemla would be produced m the rat under study m the former case, and pooled blood would undergo hemolysis as well as containing higher levels of vasoactwe blood-borne horm o n e s hke vasopressm A cumulative blood volume introduced into the reclprant rat through the cross-circulation was approximately 5 ml a rat, provided that blood flow was determined 4 times m each experiment While multtple blood groups exist m rats 7, apparent a b n o r m a l hemodynamlc reactmns m the rats or hemolysis of the reference blood samples, which might occur due to blood group incompatibility, could have never been observed Each donor rat, which had been given the same large a m o u n t of blood m each

experiment and employed m several experiments undertaken two days apart over 1 week, did not reveal behavioral or dermal a b n o r m a l m e s or respiratory &stress As reported previously by Sadoshmaa et al 9, we confirmed that w~th the tnjectlon of a large dose ot spheres (130 x 103 beads), multiple m e a s u r e m e n t s of the brain blood flow and of the responsweness of the flow to hypercapma were adequately reproducible No neurological disturbances were noUced in studies with conscious rats when 300 x 103, or even 1,440 x 10~ mmrospheres had accumulated n 12 It has been reported m a conscious dog that mlecUons of a cumulatwe dose of 24,800 x 10~ mlcrospheres (eqmvalent to approximately 250 × 10 ~ to 410 × 10~ beads a rat) did not lead to any neurological l m p m r m e n t 5 Nevertheless, we attempted m the present study to reject a smaller dose of spheres (48 x 10 ~ beads for each d e t e r m i n a t i o n of the blood flow), m order to lessen an a m o u n t of formgn bodies hke mmrospheres in the brain clrculauon We found that m j e c u o n of this small dose &d not lead to slgmficantly different results from those o b t a m e d with the large dose This held true even within such a troy region of Ussue as the hypothalamus m whmh only several tens of beads were lodged at each mjecUon The accuracy of m e a s u r e m e n t with such a small n u m b e r of spheres could still be improved by a large n u m b e r of experiments ~ and by longer counting umes for d e t e r m m m g the radloactiv~ty ~ U n d e r normocapma. 500 beads and 207 beads were lodged within the enUre bulk of the coruces and that of lower bramstem respectively These n u m b e r s were considered to be sufficmnt to permit accurate m e a s u r e m e n t of the blood flow ~ ~ 4 More detaded and sufficiently accurate determ m a t m n of blood flow of regions within the cerebral cortex may therefore be feasible after undertaking cortical parcellauon

171

1 Buckberg, G D Luck J ( , Payne D B , H o f f m a n J I E Archle, J P and Filler, D E Some sources of error m m e a s u r i n g regional blood flow with radioactive mlcrospheres, J Appl Phystol, 31 (1971)598-6(14 2 De Le~, G Nshlmvumurem'vl J - B and Leusen, I Hemispheric blood flov, in the rat after unilateral c o m m o n carottd occlusion evolutton with time, ftrol, e, 16 (19851 69-73 Hfllerdal M Sperber G O and Bill, A , The mlcrosphere m e t h o d lot measuring low blood flows theory and computer simulations applied to findings m the rat cochlea, Acta Ph~ ftol Scand, 130 (1987) 229-235 4 Ishlse, S Pegram B L Y a m a m o t o , J Kltamura, Y and Frohhch E D , Reference sample mlcrosphere m e t h o d cardmc output and blood flows m conscious rat A m J Phvs:ol, 239 (1980) H 4 4 3 - H 4 4 9 S Marcus, M L Helstad D D Ehrhardt, J C and A b b o u d , F M Total and regional cerebral blood flow m e a s u r e m e n t ~mth 7- 10-, 15- 25- and 50-/~m mlcrospheres J Appl Phv~:ol 40 (1976) 501-5(17 6 NakaL M Y a m a n e ~ , U m e d a Y , Inada M , Y a m a m o t o J and K a w a m u r a M A b s e n t effect of plasma vasopressm on rat brain blood flow during h e m o r r h a g e Am J Phwlol, 257 (19891

in press 7 0 w e n , R D , Earlier studies of blood groups m the rat Ann N Y Acad fct 97 (1962) 37-42 8 Pannier, J L and L e u s e n , I C~rculauon to the brain of the rat during acute and prolonged respiratory changes in the acid-base balance Pflugers Arch , 338 (1973) 347-359 9 Sadoshlma S B u s q a D W and Helstad D D M e c h a m s m s of protection against stroke m stroke-prone spontaneously hypertensive rats, A m J Ph~slol, 244 (198"~) H 4 0 6 - H 4 1 2 10 Solomon, R A Antunes, J L Chen YZ Bland L B and Chlen, S Decrease m cerebral blood flow m rats alter experimental subarachnotd h e m o r r h a g e a n e t animal model, ~troke 16 (1985) 58-64 11 Stanek K A Smith T L M u r p h y W R and C o l e m a n T G H e m o d y n a m l c d~sturbances m the rat as a funcuon of the n u m b e r of mlcrospheres injected A m J Ph~tol 245 (1983) H920-H923 12 Tsuchlva M Walsh G M and Frohhch E D , Systemic h e m o d v n a m l c effects of mlcrospheres in conscious rats A m J P h ~ l o l , 233 (1977) H617-H621 13 Wallenstem S Zucker C L and Flelss J L Some staUsUcal m e t h o d s useful in clrculaUon research C~r( Re~ 47 (19801 l - 9