Fluorocarbon perfusion of the isolated rat brain: measurement of tissue spaces, EEG and oxygen uptake

4~6

B[ochimica el Biopk~3A.u Aura. 1073 (1991) 486-492 ~©1991 Elsevier Science Publishers B.V. 9304-4165/91/$03.50 ADONIS 0304416.~9t00123G

BBAGEN 23477

Fluorocarbon perfusion of the isolated rat brain: measurement of tissue spaces, EEG and oxygen uptake S t e p h e n A . K . H a r v e y ~, M i c h e l e L. T r a n k i n a 1. , , M e r l e S. O l s o a ~ a n d J o h n B. Clark 2... Department of t3ioeflemisrry. University of T e x ~ Heotth Science Center, San Antonio, TX (U.S.A.) and. " Departmem of BJochemislry. St. B~rtholoote~,~ Hospital Medical College, University of Landoa, London (O.K.)

(Received11 September1990)

Key words: Per!used brain; Tissu~ space; Ele~tro~ncephalogram; Oxygen consumption; (Rat)

Previously, we have used the isolated pedused rat brain (IP]R~) m demonstrate authentic cerebral synthesis of tim lipid mediator platelct-amtivating-factor (Kumar, R., Harvey, S.A.K., Kaster, M., Hanohan, D.J. and Oisoa, M.S. ( 1 9 ~ ) Biochim. Biophys. Aeta 963, 3?5-383). The present study demonstrates that this fluorocarbon pedasion technique maintains the integrily of the blood.brain barrier (BBB), as evidenced by the small vohmt¢ (1.77-3.33%) accessible to [carboxyl.~4C]inulin. 51-66% of the brain was accessible to 3HzO, except for the spinal cord which is poorly peffused (16% accessible to 3HzO). There is no effective per[uslon of muscle l~sue associated with the ~ f i o n ( < 6% accessible to 3H 20), Fast Fourier Transform analysis of digitized EEG data showed that in low |reqnency b~md$ ( < 7.5 Hz) the IPRB had reduced electrical activity relative to the whole conscious a n i m a l The GABA antagonist bieucalline, which h ~ convulsant effects ia vivo, causes a 3-,I-Iold increase in overall (root-mean-square) electrical activity, but decreases further the relative amplitude of low frequencies. With appropriate corrections, measmmncot of l i e oxygen consumption of the IPRB can be made without the necessity for venous cannulafion. Oxygen consumption of the IPRB is flow-dependent. At a perfusion rate of !.54 m l / m i n per g, unstimulatod oxygen consumption o[ the IPRB is 2,07-2.23 t~mol/min per g, or 67-72% of the consumption of the brain in vim. Administration of bicucollino to the IPRB causes a 31% increase in lactate c411nx, but only a 15% increase in oxygen uptake, ~ggesting that tim l~mlmration becomes functionally ischemic. Measurement ol A T P / A D P levels in control and bietmulline-treated I P l U ~ otmfirms this. Other vmrkers hme used the IPRB as a model for the cerebral effecls of pharmacological agents ~ of nmtak.lic insult. The present study shows that under various experimental conditions oxygen uptake, analytical EEG measurements, and the inte~'ity of the blood-brain barrier all can be monitored.

Introduction In 1967. Andjus c t a l . [1] reported a method [or vascular isolation of the rat brain, and this isolated per!used rat brain (IPRB) preparation has proved suitable for long-term experiments [2]. 'Simplified blood',

" Present address: Department of Biology, St. Mary's LlnlvcrsiL'/, San Antonio. TX 78294-0499, U.S.A.

• * Present address: Departmentol Neurachemistry,The Institute of Neurolog 7, Queen Sqmue, London WC1N 3BC,, U.K. AhP,reviat[ons: IPRB, isolated pcrfused ral brain; EEG, electroencephalogram: BBB. blood-braln barrier. Correspondence; $,A.K. Ha~ey. Department o[ Biochemistry, University of T~xas Health Science Ccnler. San Antonio~ TX 78284-7760. U,S.A.

physiological saline containing washed erythrocytes, was used in these and other studies [3,4]. In more recent work, this perfusion medium has been superseded by chemically-defined fluorocarbon (FC)-contalning media, which have been used succcss[ully as blood substitutes in the whole rat [5] and in the isolated per!used rat brain [6.7]. Morphological studies indicate that thu blood-brain barrier (BBB) of the IPRB is intact if buffered saline is used as the perfusion medium [8]. However, to our knowledge there has been no assessmcnt o[ BBB integrily in the fluorocarbon-per!used rat brain. The fluorocarbon-per!used rat brain has been used 1o assess the effects of aglyccmla [9] and ano~a [10] on the cerebral content of adenine nucleotldes and glycolytic int~mediates. In this type of study, the IPRB is most conveniently used with the venous effluent uncannu-

497 luted. Oxygen consumption is thought to be a better indicator of survival of the perfused brain than glucose uptake [11]. Oxygen consumption measurements are best made using a cannulated effluent uncontaminated by contact with the atmosphere. To resolve this problem, we have used radiotracer measurement of experimental compartment volumes to correct ozygen consumption data acquired in an open (uncannulated effluent) system. Experimental procedures

Materials Fluorocarbon perfusion medium (FC-43) manufactured by Green Cross, Osaka, Japan was purchased from Alpha Therapeutics (Los Angeles, CA). FC-43 is a Krcbs-gingcr bicarbonate medium containing 10 mM glucose, 20~ ( w / v ) perfluorotributylamine emulsion as an oxygen carrier, 2,56% ( w / v ) Pluronic F-68 surfactant as emulsion stabilizer, and 3.0% hydroxyetliyl starch as oncotic support. At 2 5 ° C , FC-43 has an total oxygen content 2.42-times that of water (Green Cross Corporation technical data), equivalent to 2.88 mM when equilibrated with 95,% oxygen. [carboxyl-laqlnulin (No. 11086, 3 ~ C i / m g ) was purchased from ICN (Irvirte, CA). ~tH20 (NET (gliB. 1 mCi,/g) was from New England N u c l e a c / D u P u n t (Boston, MA).

Methods Preparation of IPRB. Male Sprague-Dawley rats weighing 220-360 g were used in these: studies. All animals received water and standard rat chow ad libiturn. and were anesthetized by intraperitoneal injections of .sodium pentobarbltal (up to 100 mg per kg body weight, in 25 mg doses). Surgery was performed essentially as described by Andjus e t a l . [1]. Heparin (1000 units per kg body weight) was injected into the jugular vein prior to cannulation of the external carotid arteries. After bilateral carotid perfusion was established, the spine and vertebral arteries were lighted and the head separated by transection caudal to this ligature. Perfusion conditions. Constant flow nonrecireu[ating peffusien was maintained using a LKB 2120 Varioperpe~ II pump. For technical convenience, we chose, like 81oviter and co-workers (e.g., g e l 12), to perform perfusions at room temperature. Perfusion medium at 24 + I * C was passed through a membrane oxygenator of the type descrlb0d by Scholz et at. [131. Si!ustic 602-235 tubing (25 feet total length) was wound in a single layer around an aluminum cylinder, which was contained in a plastic jacket through which a mixture of 95~ O ~ / 5 ~ CO2 was passed. The preparation was supported on a 1.5 mm stainless steel mesh on top of a polyethylene funnel (7 cm diameter x 8 cm height). A Clark-type oxygen electrode was sealed to the stern of the funnel, and perfusion medium flowed over the electrode before exiting to a fraction collector. Medium in the reservoir

was mixed by magnetic stirrer. Pressure ,.vithin the perfusion system was measured with a Statham P23D transducer, linked to a Grass 7P122C amplifier. All studies were preceded by a minimum of 20 min perfusiva to wash blood from the preparation. Radioisotopes (dissolved in FC-43) vcere infused unilaterally into a perfusion cannula using a buuerfly cannnla a n d syringe pump. Bic~qulline was added directly to the perfusion fluid reservoir. Lactate was measured enzymaticaUy t14] in the aqueous phase of the perfusate, after centfifugation (9800 x g for t5 rain at 4 ° C ) to remove the fluorocarbon, Analysis of electrical activity of the IPRB. After completion of vascular surgery a 20 gauge needle was used to bore holes in the skull over the parietal cortex. A bipolar electroencephalogram (EEG) was recorded from electrodes placed in these holes and fixed ~-mg dental cement, The electrodes were made of a 5 - 7 mm length of 26 gauge platinum wire soldered to a brass connector. Silastic or polypropylene s l a v e s ensured that onty 1-1.5 mm of the electrode wire was beneath the surface of the skull, so that direct contact with the surface of the cortex was avoided. The EEG was amplified using a Grass P S l l J amplifier and displayed using a pen recorder, in some experiment~, amplified EEG signals were digitized at a sampling rate of 250 Hr., using a Dash 16 analog-to-digital conversion card (Mefrahyte Corporation, Taunton, MA) and an IBM PC-type computer. 100 epochs of 2048 samples (each epoch = 8.192 s) were collected at evenly spaced intervals throughout the perfusion period. Digitized data were stored on 5.25 inch diskettes, then analyzed off-line using software similar to that of Webber [15], employing the Fast Fourier Transform ( F F r ) algorithm of Cooley and Tukey I161. Direct measurement of~ tissue spaces. After 50 min perfasion, a mixture of H o e and [~4C]inulin was infused for 10 min to ensure equilibration of the radioisotopes with the tissue. As rapidly as possible following this infusion the Iperfusion lines were removed, the skull opened and the brain removed and dissected. Dissected brain regions and muscle pieces were rapidly weighed and placed in 2 ml aliquots of Beckman BTS-450 tissue solubilizer, in tightly capped scintillation vials. Following solubilization (24-48 h), scintillation fluid was added and the amount of each isotope determined using a dual-channel scintillation counter, Measurement of oxygen uptake. Prior to each experiment, the oxygen electrode was calibrated at the desired experimental flow rate ( F m l / m i n ) using air to give baseline deflection and 95% 02 to give a deflection of baseline plus D ( F ) ; respectively. Now D ( F ) is giverz by an equation of the form:

D( F I = A. fl -exp(b/F)).expl - c/F I where A, b a n d c are constants.

(I)

488 15

1

~o

ues o f these constants were determined in separate calibrations where thc flow rate w a s varied (see Fig. 1). Student's t-test w a s used to determine the statistical significance of data.

1c

Results

i

Extent of perfusion of, and integrity of B B B fn, the I P R B

¥ Fiery' rme (~)r I~1/1~ Fig. I, Oxygen electr.,>de calibration was carried out as described in axe Experimemal procedures. A weighted plaStic dish trapped the canrtulae on Ihe support grid, Ele0ectlnns at different flow rates were expressed relative to deflection given by a flow rate of 160 ml/h (2.66 trd/mia) dLtriug the ~ame calibration, IsI and vertical bars represent the mgan ~ S.E, [or the number of calibrations indicated. Ii~ a separate calibration, vafialion of flaw by smaller increments over a wider range showed that this was indeed the shape of the electrode respon~ curve. The Create rcprc,~nts a least-squares fit of the Eqia. y=N.(1-exp( b/F)).exp(- c/F), giving N = 2.85, b = 3.64, and c -1.98. N is a normalization constant, produced by setting the deflation at 160 ml/h to unity. For oxygen uptake raeasurcmonts, the funnel vniumu (1:10 nil) wo~, increased by 500 FI to aeeommedat~ the, de¢trode. Since the ~rrc¢tioa cons~nt~ are proportiov,al to volun~e, the CUrreeted value of c is 1.98.(1.41/(1.70 + 0.50)~ =].27.

( 1 - e x p ( b / F ) ) is the fractional efficiency o[ the m e m b r a n e oxygenator and e x p ( - c / F ) ) is the fractional recovery of oxygen at the electrode assembly. T h e val-

while: the BBB has b e e n reported to be intact j a preparations perfused with erythrocyte-containing m e d i u m [17] o r with saline [8], the possibility w a s explored that the fluorocarbon m e d i u m used in these studies m i g h t alter the permeability of the barrier. Regional weights w e r e c o m p a r a b l e with those previously published for r a t b r a i n rapidly isolated f r o m the whole a n i m a l [18], s h o w i n g that there are no gross c h a n g e s in weight o f the I P R B d u e to e d e m a or dehydration. T h e w e i g h t o f b r a i n which is efficiently perfused is t a k e n as 1.73 g ( s u m o f m e a n regional weights f r o m T a b l e 1, excluding the spinal cord). M e a s u r e m e n t s were m a d e of the [14C]ioulin a n d 3H~O spaces o f the p r e p a r a t i o n by unilateral infusion o f these radioehemieals (Table 1). T h e a p p e a r a n c e of tracer in the contralateral zegions of the b r a i n indicated c x d r a n g e via the Circle of willis. T a b l e It shows the actual vascular volumes a n d fractional e x c h a n g e rates calculated f r o m d a t a in T a b l e L Vascular v o l u m e s a r e c o m p a r a b l e w i t h values reported for unilaterally p e r f u s e d b r a i n in vivo [19], a n d for values in whole r a t I201. T h e relative w a t e r spaces o f tissue samples reflect line relative efficiency o f perfusion. T h e spinal cord is considerably less well perfused than other brain regions, as noted previously [17]. I n m a p l e samples the [t4Ctinulin

TABLE I

~Yeight, [ C/tnuttnspace, and t1#0 space in different regionsof the IPRB IPRBs were perfused, dissected ~md weighed as described in E.~perlmetttei proeoflures. Brlfiu regions were sampled whole- Sobeortex inetudc:d ( ~ nef. 18) the mld,brahh thalamlc rcgloas and (because of the speed of dissection) varlab]e amounts of the klppocampus and corpus callosum, partial ~mptes of muscle td~ were taken. Vaiu¢s are means ± standard deviations for the following numbers of determinations: weight, seven inulin space four, water space live, SAmples ',ver¢solabilize.6, then sttlojcctcd to dunl-¢h~tl¢l scilltilintioa coontirtg writ ©sternal quoth m c a s t t ~ t , Separate .~is of 3H~O and [14Clinulia standards w~e counted no.din the same quench conditions, and the isotope contents of each sampls calculated. The % [raeur spare for each sample wa~ calculated as: (lO0,O.8,(d,p.m,/m$ of tissue))/(d.p.m/p.I perfuslon fluid). The racier of 0,g appears Jn the numerator since this is the fraction of perfusion fluid which is aqueous (see Experimental procedures), and therefore available to hydrophilic tracers. Weight (rag) meaa

[14C]lnulin sxoace(g)

~H~O space (~)

$.D.

mcun

S.D.

mean

S.D.

It. cortes

426

37

1.35

0.50

55.25

] 1.22

R. subeottcx L, cortex L subeor tes Cerebellum M.O.P, Spinal cord Neck muscle R. mt~sclc L. muscle

209 454 Xl0 267 lg3 112 572 291 283

3I)

2.27 U,42 0.76 1.67 1.42 o.'~i 0,60 0.61 0.60

0.99 0.13 0.14 0.44 0.18 ~,2~ 0,32 0,27 0,25

57.34 6,77

10,82 2.71 3.36 8.77 ~.98 2.45 2.46 1.32 2.57

33

48 32 19 29 200 107 ~

g.43

30.65 25.55 7.84 2.69 2,11 2,72

489

TABLE lI Compori~on between ~eacular spaces m the ~*olatad per[used rot brain oad r~ose in the rat brain in ~ilu

For cortex and sab~ortex, if V, is the apparent volume of the ipsilateral (R~H.S) re#or and V~ the apparent volume of the contralatera[ (L.H.S) region men the actual tracer volume of the ipsilaletal region will eqcal (Vii+ V~) and the fractional exchange will eq~sl {V~)/{V, + VD. Table II shows these actual volsmes and fractional-exchange rat~s, calculated from the moan values in Table I, Regaon

Space (~) Fraetional Range of c~r~hral va-~uhir inulin water exchange space (~.) in whole rat.

Cortex t.77 Subcormx 3.02 Cerebellum 3.33 M.O.P 2.83 Spinalcord 1.8~.

61.9 65.7 61.2 51,1 16,1

0.235 0.2,51 (0.5 *) (0.5 *) (0.5 *)

1.58-1.70~ 2.Dg-z42 ~ l.]0d.40 ~ 1.24-26.82 b~ 3.46 t, 2,56-3. 53 b

* The fractional exchange for cer~beltum, medulla obtongata/ports and spinal cord is O.S by definition, as thes* regtons are equally pcffused by hath cammla¢. In these regions therefore., actual spac~ (as shoo,an here) is d0ubl¢ ~h¢ ~po.rcnt space shown in Table l, u Values l'mm PmC 19. Values from Ref. 20, " The BBB is ab~la in the pituitary, which cons~!uently l~as a high apparent va_culat space_

a n d 3HaO spaces are v e r y similar, showing that, while there m a y have been c o n t a m i n a t i o n or passive infiltration of m e d i u m into t h ¢ ~ regions, continuous perfusion did not occur. In s u m m a r y , the low values for the brain [~C]inulin spaces d e m o n s t r a t e that the BBB is intact, while the t~lative values for s H o O spaces c o n f i r m that the brain is the principal site of pcrfusiou of the preparation.

Use o f compartment oxygen uptal
volume measurements

to correct

W e showed p~eviot~sly [21] by indirect measurements that the [~4C]inufia-acc~ssible space in the I P R B is m u c h smaller than the aH~O-aceessibl¢ space. T h e efflu~ curves for ['~C]inulm [21] can be used to calculate that the v o l u m e o f IPRB-plus-appatatu~ is 3.11 + 0.51 ml ( m e a n 4-S.D., n = 5). W i t h th~ a p p a r a t u s alone, similar measureraents yielded a volume o f 1,70 _+ 0.23 ml (data not shown). T h e vascular space of the I P R B is small ( < 0.1 lift: Table !), so the difference between 1PRB-plas-apparatus a n d apparatus alone (1.41 nil) represents an additional volume which pools under the preparation. T h i s v o l u m e incurs diffusiv~ loss of oxygen a n d is used to correct the constant c for the calculation o f oxygen uptake (see legend to Fig. l). In each experiment conducted at flow rate F m l / m i n with a calibration deflection of D ( F ) , the corrected 0xvgen c o n s u m p t i o n ( o m o l / m i n per g) was calculated f r o m the time-d~pendent experimental deflections d ( F ) ~. (l

l

eXp(b / F ))" (I -- (¢xp(l "27/F ))" d ( F ) / D ( F ))

(2)

W h e r e b has the value (3.64) determin¢d b y calibration a a d 1.27 is th~ ~ r r e c t g d value o f c (se~ l~gend to Fig. l). A is given b y ( F . K . O . 7 4 / I V ) , whero K (2.88 r a M : G r e e n Cross Technical D a t a ) is the total concentration of o x y g e n in FC-43 which is in equilibrium with 9 5 ~ O 2 / 5 ~ CO~, a n d W is the perfused weight of the brain. T h e factor 0.74 a p p e a r s since w e have defined full-scale deflection as 9 5 ~ 0 2 to 2 1 5 On, therefore only ( 9 5 - 2 1 ) / ( 1 0 0 - O) o f the full o x y g e n content of the FC-43 is available.

25] I

a~

<

2

2

1.5.

14 1

EEG

Fig. 2. [n three of the experiments de~crlhed in Fig. 3, the large mcreasu in eleslrical a~tivity routinely sccn on infusion with b~euculline was quan~fied as described in Experimental proctors, Data was collected for 12 mia intervals. Fr~lUCn~.'yImnds m~ a~ defined in Rcf. 22:D1 (0.|-L4g l-lz) and I)2 (L56-3.51 Hz) are ~ b.nd~ TI (3.61-~,57 l-lz) and 3"2 (5.66-7.52 1ix) an: 0 bands. At (7.62-9.47 Hz) and A2 (9.57-12.50 FIg)are a bands, BI (12.60-1"/.48Hz) and ~7. (17.58-25.0 Hz) ~¢ fl hands. Ca) Shows the amplitude in ~i'fereatfrequency bands for data colIecmd during the periods (mill):0-12 (I), 13-24 ([3),25--36 (~-)and 37-48 (+), (b) Shows data for 0--48 rain uv¢cag~l to~thgt (I),in gompafi;sonwith data obtained from the whole conscious rat (a), Data For the period (49 60 rain} of bicugulline stimulation is shoWr~separately Kl).

490 Eleezrieal responses of the IPRB in vivo, cerebral electrical activity is an important indicator of neurological function. We routinely recorded the electrical activity of the IPRBs. The magnitude of this activi,'2, could be increased by bicuculline v, ith typical "spiking" of the signal [1]. Fourier transformed data (i.e., amplitude vs. frequency) was averaged within the frequency bandwidths used by others [22]. Fig. 2a shows the activity of the preparation in sequential 12 rain periods during perfusion_ Some loss of low frequency signal (i.e.. in 8 and 0 bands) occurs after the first 12 min. The amplitudes of a and fl bands are little altered throughout the perfusJon period. In Fig. 2b, the data from Fig. 2a has been averaged together and is compared with data collected by others [22] from the whole conscious rat. Rdative to the whole animal, the IPRB shows decreased ~- and 0-band activity, particularly 8~ and 0~. In contrast, the a- and/~-band amplitudes are somewhat higher than data from the whole animal. For the final 12 rain period where bicueulliaeinduced 'spiking' occurred, the apparent amplitude of all band~ is inoreased. However, the electrical activity in the # bands shows a decrease relative to the rest of the power spectrum.

Response of oxygen consumption anal lactate output to bicuculline treatment and to altered flow rate Oxygen electrode deflection increased significantly ( P < 0.04) between 1-6 rain of perfusioa (Fig. 3). This demonstrates a decrease in oxygen consumption, probably due to cooling of the IPRB from 3"/ to 25~C. There was a further significant ( P < 0.05) increase in

0

10

20 30 P~rlt,l~lOn tl['~e (rain)

40

50

6£")

Fig, 3, Shows the oxygen electrode deflect/on (12,} for ¢~g:~hl experiments where bicueullio¢ was used to stimulate cerebral melaholism. Perfusion flow rate for these experimenls was 2.66 m l / m i a (160

ml/h), Solid line is the envelopeof Ihe mean-$.E, values. Numbers indicate the pettusion time~ (i,~ minut¢~) f o r which absolute rates of oxygen COnsumption w~e calculated (see i~xt), In the calibrations for

these experiments,baseline(equilibraledwilh airl gavea defleclionof 74d-2~, and [baseline+D{F)I (equilibratedwith 95~ O2/5~ 002) of 226± 23~ (mean±S.E.).

25,

/ A

'~

[

05

°(4

te

~e

2 22 24 f f l ~ rale, ml,'m~n

25

28

Fig. 4~ Shews the oxygen uptake ([3) and lactate output {A} of preparations perfumed at different flow rates. Measurements were made after at 20-25 rain peffusion, i,e,, after the initial decrease in

both parameters sccn in Fig. 3. Each preparation was per[used at a single flow rate. La~Iecl points are the m~ans of ¢h¢ O u m b © r ol separate preparations indicated, Regression tines were calculated usina

individualvalues.

deflection between 6-44 rain. Infusion of bieuculhne caused characteristic spiking of the EEG from 46 rain, accompanied by a significant (P 0.05).

Diseusslon The 1PRB can he manipulated experimentally to explore cerebral metabolism without attendant systemic complications. In order to employ the IPRB with confidence, the preparation must meet three criteria: there must an intact eBB, maintenance of electrical activity,

491 and appropriate metabolic activity_ The characteristics of uptake of glucose and 3-hydroxybutyrate by the 1PRB [3,4] and the inability of pyravate to maintain the preparation ( g e l 7 and Harvey. S.A.K. and Olson, M,:S., unpublished data) are consistent with the BBB being intact. However, to our knowledge this is the first direct measurement of vascular spaces in this type of preparation. It has been reported [1] that the EEG of the isolated perfused rat brain is very much like that of the intact hypothermie tat of the sa~ne temperature. The rootmean-square voltage for the present preparation is 29 #V, wl'dch is comparable to the value of 47/~V found for the IPRB maintained at 37~C by another laborator3, [6], taking into account the lower temperature of the present preparation. Other workers have performed frequency analysis of EEG recorded from the fluorocarbon-perfused rat brain, using the baseline crossing technique of Sahzburg e t a l . [23]. We have quantified more precisely the EEG output of the fluorocarbon-perfused rat brain by using the Fast Fourier Transform (FFT) method. The pemobarbital anesthesia used during the preparation of the IPRB decreased the amplitudes of low frequency brads relative to the equivalent frequencies recorded from whore conscious rats [22]. After the first 12 rain, cooling oF the preparation results in a further decrease in ~- and 0-band amplitudes. It is during this period that decreased oxygen consumption and lactate output also are apparent. Meldrum and Nilsson [24] reported a value of 7.6 ml O2/100 g per rain for the rate of oxygen consumption of rat brain in rico. This rate of oxygen consumption (equivalent to 3.1 p m o ! / g per mln) in ~omcwhat Ligher than the unstimulated oxygen uptake (2,07-2.23 t~mol/g per rain) of the IPRB at 25"C, due probably to the temperature difference, In the IPRII there is a linear relationship between oxygen uptake and flow rate, similar to that found by others [11] for the dog brain perfused at 37°C, The regression equation for oxygen consumption from Fig. 4 is Y = 0,47 + 0.94- X, if the x-ar..is is converted to specific flow ( m l / m l n per g). Recalculation of data from Ref. 11 to provide the same units gives an Eqn, Y = 0.50 + 1.61. X, Thus, the intercepts (extrapolation to zero flow) for these two sets of data are very similar. In rico, the specific oxygen consumption of smaller animals tends to be higher, therefore the intercept for the 1PRB might be expected to be higher than that for the canine preparation, The lower than expected value for the intercept, and the (1.71-fold) shallower gradient for the IPRB relative to the canine brain probably are due to the lower temperatttre of the IPRB in this study. It was reported [1] that the convulsant MetrazoI (pentylenetelrazoi) increased lactate output concomitant with increased electrical activity in the IPRB perfused by reeirenlating "simplified blood'. We have con-

Firmed and extended these findings by performing similar experiments using an erythrocyte-free aonrecireulating system and have observed also art 15~ increase in oxygen consumption upon the administration of bicueulline. In vivo, the effects of bicuculline (i.e,, 2-3-fold increase in both oxygen consumption and glucose utilization) are assooated with a massive increo,s¢ in cerebral blood flow (3-5-fold: Ref. 24). Under conditions of constant flow, these metabolic changes in the IPRB axe attenuated. The implication of this observation is that blcueuliine stimulation of the constant-flow IPRB results in functional ischemia. The A T P / A D P ratio of preparations treated with bieueulline (3.40 + 0.70. mean + S.D., n = 7 ) i s s~nificantly ( P < 0 . 0 O l ) lower than that of corttroI preparations (9,10 + 0.76, mean +__S.D., n - 8) confirming that this is the ease. With the exception of the perfu'_-ed dog brain [251 perfused brain preparations erdtibit rates of lactate elflux higher than those found in rico. The basal lactate efflux from the present preparation i~ SOmewhat higher than that observed in IPRB by others 126], but lactate production is sufficiently low to he due to hypoxic glycolysis in a poorly" perfused region of the preparation (i.e,, the spiral cord). This observation is consistent with the lactate efflux remaining essentially constant at all flow rates (F;,g, 4), The [PRB has been used extensively to study the direct cerebral effects of pharmacological agents [27-30]. In many of these reports, the preparation is maintained at 37°C. The present study demonstrates that quantitative measurements of EEG and oxygen consumption can be made successfully, even when these parameters ore attenuated by malntcnance of the preparation at a lower temperature, The [PRB also may be used to confirm the cerebral origin of endogenous mediators, Bicucullingqaduced seizures in the whole rat cause an increase in the cerebral content of plmelet activating factor (PAF), a potent vasoactive mediator. We have e~tablished [21] using the bicucullinc-s~t~mulated IPRB that this synthesis of PAle is authenticMly cerebral, lndlreet evidence [311 suggests that in rico, PAF plays a role also in the cerebral response to ischemia. Since the bieueuLline-stimulaled IPRB is functionally isehemie, it will he necessary to clmracterize the contribution of simple ischemin fi.e., reduced perfusion flow rate) to PAF synthesis, The ability to measure the rate of oxygen connumption of the IPRB c,Ver a range of flow rates will facilitate these studies. Aekn~hag,em~ts This research was supported by NIH grant NS 19426. The authors gratefully acknowledge Dr. V. Gene Erwin for his help in establishing the brain per(usion technique, Dr. Neat Robinson and Dr. David S. Lapointe

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