Upsala J Med Sci 92: 205-213, 1987 Positron Emission Tomography: An Animal Model of Spinal Distribution of Drugs After Intrathecal Administration Per Hartvig’, Lars L. Gustafssod, Kjell Bergstrom’, Ulf Mostrom’, Urban Ponten3, Bo Lindberg4 and Hans Lundqvist’ ‘Hospital Pharmacy, Departments of ’Diagnostic Radiology, ’Neurosurgery and 4Primate Laboratory, University Hospital, ’Department of Physical Biology, Gustaf Werner Institute, University of Uppsala, Uppsala and 6Department of Clinical Pharmacology, Huddinge University Hospital at Karolinska Institute, Huddinge, Sweden ABSTRACT An animal model has been developed in the Rhesus monkey for noninvasive monitoring of CSF transport of drugs by external detectors i.e. positron emission tomography. The model compromises the cannulation of the subarachnoid space (with a spinal needle), and has been used without any damage to the monkey. With the method it was shown that injection rate had a major influence on the transport rate of 68GaC1 in the CSF. Injection of 0.5 ml over 60 sec gave the highest radioactivity near the injection site, whereas an injection rate of this volume shortly after injection. This method have been of value for the determination of drug kinetics after spinal administration. 3 over 10 sec resulted in high radioactivity more rostrally INTRODUCTION Positron emission tomography (PET) is a noninvasive technique which measures the kinetics of biochemical processes and transport of radiolabelled compounds in vivo (5,6). The technique furnishes cross-sectional images o f the distribution o f radioactivity in the body and can be regarded as an in vivo autoradiographic method. The technique may be potentially useful in measuring cerebrospinal (CSF) bulk flow o r the distribution of low molecular radiotracers after spinal (intrathecal o r epidural) administration, since kinetics a r e monitored noninvasively by external detectors. The rostra1 bulk flow of CSF has been studied by gamma scintigraphy using radio- tracers such as iodinated human serum albumin (4). It is doubtful, however, if albumin will mirror the distribution of small drug molecules owing to differences in molecular weight and physio-chemical properties. 205 An e x p e r i m e n t a l model u s i n g Rhesus monkeys was d e v e l o p e d t o s t u d y t h e d i s t r i b u - t i o n o f i n t r a t h e c a l l y a d m i n i s t e r e d 68Ga b y PET. The o b j e c t i v e s were t o : * e v a l u a t e a model u s i n g P E T f o r t h e measurement o f t h e t r a n s p o r t o f s m a l l r a d i o l a b e l l e d m o l e c u l e s i n CSF a f t e r s p i n a l a d m i n i s t r a t i o n * s t u d y t h e i n f l u e n c e o f i n j e c t i o n r a t e on t h e d i s t r i b u t i o n o f t h e r a d i o t r a c e r i n t h e CSF. MATERIALS AND METHODS Animals Rhesus monkeys (Macaca M u l a t t a ) w e i g h i n g 6.3-10.5 k g from t h e P r i m a t e L a b o r a t o r y o f R e p r o d u c t i v e Research, Uppsala u n i v e r s i t y were used a f t e r an o v e r n i g h t f a s t . The a n i m a l s were a n e s t h e t i z e d w i t h r e p e a t e d doses o f 50-100 mg o f k e t a m i n e i n t r a - m u s c u l a r l y ( K e t a l a r , Parke-Davis NJ, USA) g i v e n e v e r y 30-60 m i n u t e s . Diazepam (DiazemulsR, K a b i - V i t r u m Stockholm, Sweden) was a d m i n i s t e r e d i n t r a v e n o u s l y as a supplement i n doses o f 5-10 mg e v e r y 30-60 m i n u t e s . A f t e r i n d u c t i o n o f a n a e s t h e s i a , t h e monkey was p l a c e d l e f t s i d e down i n a s p e c i a l l y c o n s t r u c t e d p l a s t i c c r a d l e , w h i c h was k e p t h o r i z o n t a l d u r i n g t h e e x p e r i m e n t . R I n t r a t h e c a l a d m i n i s t r a t i o n The s p i n a l c a n a l was p u n c t u r e d between t h e s p i n a l p r o c e s s e s L3-L4 w i t h a m o d i f i e d s p i n a l n e e d l e 40 mm l o n g and w i t h a dead space o f 50 p1 ( E v e r e t t s p i n a l n e e d l e R , Avons M e d i c a l L t d R e d d i t c h , England, 0.7 mm ODx90 mm). To s t a b i l i z e t h e n e e d l e d u r i n g t h e e x p e r i m e n t i t was passed t h r o u g h a p l a s t i c s t o p p e r , t h a t was f i x e d t o t h e n e e d l e w i t h a screw and t o t h e s k i n w i t h a t a p e . The escape o f one d r o p o f CSF i n d i c a t e d p u n c t u r e o f t h e s u b a r a c h n o i d space. The n e e d l e had a s h o r t b e v e l , w h i c h was p o s i t i o n e d w i t h i t s o p e n i n g p o i n t i n g c r a n i a l l y . B e f o r e t h e i n j e c t i o n o f r a d i o a c t i v i t y , t h e p o s i t i o n o f t h e n e e d l e was v e r i f i e d w i t h f l u o r o s c o p y , a f t e r t h e i n j e c t i o n o f 0.1 m l m e t r i z a m i d e (Amipaque , 170 I / m l , Nyegaard, Oslo, Norway). The CSF-pressure was m o n i t o r e d b y c o n n e c t i n g t h e s p i n a l n e e d l e t o a Gould p r e s s u r e t r a n s d u c e r v i a a s a l i n e f i l l e d p o l y e t h y l e n e c a t h e t e r . R The s i z e o f t h e s p i n a l c a n a l was c a l c u l a t e d b y computed tomography (CT; Siemens Somatom DR2) i n a monkey w e i g h i n g 9.3 kg. The c r o s s - s e c t i o n a l a r e a o f t h e s p i n a l c a n a l was e s t i m a t e d a t d i f f e r e n t l e v e l s by m e a s u r i n g t h e t w o axes i n t h e a x i a l C T scan and u s i n g t h e e l l i p s i s . The volume o f t h e s p i n a l c a n a l was c a l c u l a t e d by m u l t i p l y i n g t h e c r o s s - s e c t i o n a l a r e a w i t h t h e l e n g t h measured on t h e l o n g i t u d i n a l scan. CSF and b l o o d s a m p l i n g A second s p i n a l n e e d l e ( E v e r e t t s p i n a l n e e d l e , 0.5 mm ODx50 mm) was i n t r o d u c e d i n t o t h e s u b a r a c h n o i d space t h r o u g h a l a t e r a l p u n c t u r e a t C1-C2 l e v e l f o r CSF 206 sampling ( 1 , 9 ) , A PE 50 polyethylene catheter was connected to the end of this needle through an adapter. Blood from a peripheral vein and CSF samyles were collected up to 40 and 60 minutes after injection, respectively. Radiopharmaceuticals 68Gallium as the chloride salt ( 68Ge/ ml of 1 M hydrochloric acid. This solution was neutralized with 1 M sodium hydroxide with phenol red as indicator. 68 GaCl ) was produced in a tin dioxide open bed 3 68 Ga column (New England Nuclear Medicine, Boston, USA) eluted with a few Before administration, the solution was filtered through a 0.22 pn membrane filter for sterilization. The radioactive dose varied between 10 and 60 MBq in a 0.5 ml sample. The injections were given in the same animal following each other (with an 2-hour interval) and given at a constant rate by a syringe pump (Model 220 Sage Instruments) over 10 o r 60 sec, respecti;ely (Table 1 ) . Table 1. Maximal radioactive uptake in the spinal canal after intrathecal administration at L3-L4. Registration with positron emission tomography. Exp Radiotracer Duration of Spinal level Maximal Time aftet. injection * injection (sec) uptake min 1 1 15 300 8 580 27 80 20 2 68GaC13 10 350 6 1 68GaC13 60 f: L 6 166 26 3 'Uptake = nCi/cm Dose x bodyweight-' (1 ) Instrumental For the imaging of different spinal levels, the cradle was moved to radio- logically predetermined positions. Imaging o f the monkey was performed at the spinal levels L6, L1 and T8 in a PC 384-38 positron tomograph (Scanditronic Instrument AB, Uppsala, Sweden) equipped with two rings of detectors that gave three simultaneous slices of 13 mm thickness each (5). Images were recorded for 60 sec throughout the study with the initial kinetics o f radioactivity monitored at the L 1 level. 207 The influence o f the injection rate was studied by using two collimated beta- scintillation detectors ( B G O ) positioned at L6 and L1 and with the positron camera at the T8 level. The distribution of radioactivity could thus be monitored at three different levels simultaneously. Calculations The radioactivity per cm3 was calculated as a function of time at each spinal level corresponding to L6, L1 and T8 and was corrected for physical decay of the radionuclide from the time of administration. The values were divided by the amount of radioactivity given per gram body weight. A normalized uptake of 1.0 corresponded to the uptake that would have been achieved if the administered radioactivity was equally distributed in the whole body of the monkey assuming 1 cm is equal to 1 9. The standard deviation of measured radioactivity was always below 15%. Radioactivity in blood and CSF were measured in a well-counter and expressed as uptake values as described above. 3 . RESULTS Animal model Intrathecal drug administration, CSF-sampling, anesthesia and positioning of the cradle in the positron camera were tested in 4 animals before the experiments started. Satisfactory anesthesia of the monkeys was achieved with ketamine and diazepam with no signs of involuntary movements o r hyperventilation. The animals could be kept in the same position f o r several hours and they did not show neurological sequela. The intrathecal positioning o f the needle was crucial, since a constant CSF- flow did not ensure a correctly placed needle. This may also occur when the needle is partly localized in the subdural space after disconnection of the arachnoidea ( 8 ) . Puncture of the subarachnoidal space at C1-C2 could be carried out without problems and samples o f 100 p o l o f CSF were taken every 15 min. The frontal and sagittal diameters at all levels of the spinal canal were less than 8 and 6 mm, respectively (Table 2). The estimated volume of the dural sac was 7-9 ml. 208 T a b l e 2. Anatomic d i m e n s i o n s o f t h e s p i n a l c a n a l c a l c u l a t e d from C T scans i n a Rhesus monkey ( w e i g h t 9.3 k g ) . L e n g t h o f s p i n a l d u r a l sac ( f o r a m e n magnum - S 2 ) : 350 mm. E s t i m a t e d volume o f t h e s p i n a l c a n a l ( f o r a m e n magnum - S 2 ) : 7-9 ml. D i s t a n c e between t h e p u n c t u r e s i t e (L4-L5) and d i f f e r e n t v e r t e b r a e l e v e l s : L7: 50 mm; L1: 75 mm; T8: 170 mm; C7: 270 mm. V e r t e b r a l l e v e l F r o n t a l and s a g i t t a l d i a m e t e r s (mm) C r o s s e c t i o n a l a r e a ( e l l i p t i c ) n c5 T4 T I 1 L1 L3 L5 6.7 x 4.7 4.0 x 3.7 5:3 x 3.7 6 . 1 x 5.3 6.7 x 5.3 7.5 x 5.3 25 12 15 28 2 8 30 P o s i t r o n e m i s s i o n tomography I n j e c t i o n r a t e h a d a pronounced i n f l u e n c e on t h e s p r e a d o f g a l l i u m ( T a b l e 1 ) and t h e model was t e s t e d b y u s i n g measurements o f t h e CSF-pressure and e x t e r n a l m o n i t o r i n g o f r a d i o a c t i v i t y a t s e v e r a l l e v e l s by c o l l i m a t e d d e t e c t o r s . The d i s t r i b u t i o n o f r a d i o a c t i v i t y a t d i f f e r e n t s p i n a l l e v e l s a f t e r i n j e c t i o n t i m e s o f 60 and 10 sec is shown i n F i g . 1 and 2, r e s p e c t i v e l y . I n j e c t i o n o v e r 60 sec gave a l i n e a r i n c r e a s e o f a c t i v i t y a t a l l r e c o r d i n g s i t e s s t a r t i n g i m m e d i a t e l y and c o n t i n u i n g d u r i n g t h e whole i n j e c t i o n p e r i o d ( F i g . 1 ) . The i n c r e a s e i s s t e e p a t L6, i . e . 1 cm c a u d a l t o t h e i n j e c t i o n s i t e . A t T8, 15 c m f r o m t h e i n j e c t i o n s i t e , t h e i n c r e a s e i s s m a l l e r b u t l i n e a r o v e r t h e whole p e r i o d . A f t e r end o f t h e i n j e c t i o n no f u r t h e r i n c r e a s e i n t h e r a d i o a c t i v i t y was measured a t L6. A t L1 and T8 t h e i n c r e a s e i s l i n e a r up t o 8 m i n with an a p p r o x i m a t e d o u b l i n g o f t h e a c t i v i t y w i t h i n 12 and 6 m i n , r e s p e c t i v e l y . B e f o r e i n j e c t i o n t h e r e c o r d e d CSF p r e s s u r e was 1 mmHg. I t was 3 mmHg a t 2 m i n a f t e r end o f i n j e c t i o n . 209 3000- 8 2000- \ Q c 3 0 I 1 0 60 120 240 360 480 600 3000- '0 S : 2000- 3 \ 0 S z 0 0 CI 1000- 0 I A - - 0 e - I I I I I I , I 7 600 T i e after injection (seconds) Fig. 1 . Inikial kinetics o f radioactivity (counts p e r second) at vertebrae levels L 6 (0) a n d L 1 (A) monitored by external detectors BGO and radioactivity at T8 (m) registered by positron emission tomography. The dose was given intra- thecally in 0.5 r n l o f 68Ga-C1 over 60 seconds at L3-L4. 3 1 210 A different pattern is shown with a 10 sec injection (Fig. 2 ) . A fast but non- linear increase in radioactivity is seen at all recording sites. The caudally located detector at L6 close to the injection site only recorded a small fraction of the total radioactive dose. The measured radioactivity at T8 is three times higher than at L6. After the injection fluctuating value over 200 sec indicate turbulence in the system. At 200 sec a slow increase in radio- activity at T8 and a slow decrease at L6 was measured. At L1 the radioactivity increased by 5 0 per cent to constant values after 6 min. Recorded CSF pressure prior to administration was 8 mmHg. It increased rapidly to a maximal value of 2 5 mmHg and decreased then slowly. The CSF pressure was stable at 4-6 mmHg at 6 min after injection. The radioactivity in CSF samples taken from C1-C2 remained low throughout the experiment. DISCUSSION The present study demonstrates a new method to monitor drug distribution in the spinal canal and is also a new application of PET. The size of the Rhesus monkey allows the body to be freely moved in the PET but the animal is still large enough for injections o f rather large volumes (0.5 ml) into the spinal canal. The model and the detection technique were, thus, adequate for studies of distribution after intrathecal administration of the radiotracer 68Ga in the spinal canal. The rapid initial distribution after an intrathecal dose was documented with the model. The kinetics of the radioactivity could be monitored at three levels simultaneously by using two external detectors and the PET-camera. Together with the sampling o f CSF at C1-C2 a good measure was given of the distribution of radioactivity. It was documented that the injection rate higly determined the spread of radioactivity in the CSF. A considerable change of volume may occur in the spinal dural sac, which acts as a pressure reducing chamber for the cranial CSF (8). Changes in the spinal CSF space are balanced by a decreased volume of the epidural veins of the spinal canal. The measured diameters of the spinal canal (Table 2) indicates that the maximal size of the spinal dural sac can be estimated to 7-9 ml. Assuming that the spinal cord diameter is about 2 / 3 to 3 / 4 of that of the spinal canal, a spinal CSF volume of 3 - 5 ml can be calculated which is in accordance to the data of Rieselbach(l0). The injected sample ( 0 . 5 ml) thus represents 1 0 - 1 5 per cent o f the spinal CSF space, which in humans would be a slightly larger volume than used to achieve spinal anaesthesia (2). 21 1 The injected volume will displace CSF and expand the dural sac. In the lumbar I region this volume would correspond to a column of 3-4 cm and in the thoracic region to 7 - 9 cm (Table 2). Due to the direction of the bevel o f the needle the displacement of CSF will mainly be cranially. With the rapid injection surprisingly small amounts of the tracer were found even just caudal to the injection site. It can be concluded that very little expansion apparently occured in the caudal dural sac during the rapid injection with concomitant high CSF pressure. The cranial CSF space on the contrary may accomodate some 10 per cent of the spinal CSF volume by changing the blood pool by 0.5% of its intra- cranial volume. The high radioactivity observed at T8 at the end of the fast injection cannot be explained simply by displacement o f CSF. A considerable turbulence with mixing o f the sample and the CSF has to be postulated. Turbulence may also explain the early increase in activity detected at T8 during the slow injection. The distribution rates of the radioactivity that are measured during the initial minutes after intrathecal injections of this volume (0.5 ml) are therefore not representative of CSF bulk flow. This affects the distribution of the injected tracer within the spinal dural sac during at least the first 5 minutes. The anatomical dimensions of the spinal canal (cf Table 2) are smaller than can be resolved in the PET-system used (spatial resolution about 8x8~13 mm). The calculated uptake values (nCi/cm /dose/bw) are relative and do not give exact quantitative measurements ( 5 , 6 ) . Consequently the measurements from PET images represent concentrations in the spinal canal as a whole and cannot be strictly related to CSF or the spinal cord. However, in the case of 68Ga almost all radioactivity would be within the CSF because of the hydrophilicity of the ion. 3 Another limiting factor of positron emission tomography is the observation time. The positron emitting tracers have a rapid physical decay with half-lives of 68Ga and "C of 68.7 and 20.4 min, respectively. This makes prolonged studies impossible. More long-lived positron emitters like 73Se(tl 7.1 h) can, when they are available, overcome such drawbacks. 2 Despite the limitations, PET gave an in vivo quantitation of variations in concentrations at different spinal levels and made it possible to study factors which influence rostra1 transport after intrathecal application. The animal PET model including technique for cannulation of the subarachnoid space may be applied to study the transport of several types o f drugs such as e.g. opioid analgesics, in the CSF after intrathecal injection. Such studies using this 212 t e c h n i q u e is p r e s e n t e d e l s e w h e r e ( 7 ) . ACKNOWLEDGEMENTS Mrs Y l v a M y r b e r g and M r L e i f Ormegard a r e acknowledged f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e and Mrs J e a n e t t e G r u n s t e i n and Mrs G u n h i l d L a r s s o n f o r s k i l f u l t y p i n g o f t h e m a n u s c r i p t . T h i s s t u d y was s u p p o r t e d by g r a n t s f r o m t h e Swedish M e d i c a l Research C o u n c i l (3902, 7005) and t h e K a r o l i n s k a I n s t i t u t e . REFERENCES 1. Amundsen, P. & Skalpe, 1.0.: C e r v i c a l myelography w i t h a w a t e r s o l u b l e 2. Bridenbaugh, P.O. & Kennedy, W.F.: S p i n a l , s u b a r a c h n o i d n e u r a l b l o c k a d e . c o n t r a s t medium ( M e t r i z a m i d e ) . N e u r o r a d i o l . 8: 209-212, 1975. I n : N e u r a l b l o c k a d e i n c l i n i c a l a n e s t h e s i a & managements o f p a i n (M.J. Cousins & P.O. Bridenbaugh, ed) pp. 146-175. J P L i p p i n c o t t Co, P h i l a d e l p h i a , T o r o n t o , 1980. c a t h e t e r placement. A n e s t h e s i o l o g y 37: 352-353, 1972. 3. Cohen, C.A. & K a l l o s , T.: F a i l u r e o f s p i n a l a n e s t h e s i a due t o s u b d u r a l 4. d i C h i r o , G.: Movement o f t h e c e r e b r o s p i n a l f l u i d i n human b e i n g s . N a t u r e 5. E r i k s s o n , L . , Bohm, C., Wesselberg, M., B l o m q v i s t , G., L i t t o n , J., WidBn,L., Bergstrom, M., E r i k s s o n , K . & G r e i t z , T . : A f o u r r i n g p o s i t r o n camera system f o r e m i s s i o n tomography o f t h e b r a i n . IEEE Trans. N u c l e a r S c i e n c e 29: 204: 290-291, 1964. 539-543, 1982. 6. G r e i t z , T . : P o s i t r o n e m i s s i o n tomography f o r q u a n t i t a t i v e i n v i v o . S t u d i e s o f n e u r o t r a n s m i t t e r k i n e t i c s : a c r i t i c a l e v a l u a t i o n . F r o n t i e r s i n B i o - c h e m i c a l and P h a r m a c o l o g i c a l Research i n D e p r e s s i o n ( e d . E. Usdin, M. Asberg, L. B e r t i l s s o n & F. S j o q v i s t ) , pp. 69-78. Raven P r e s s , New York, 1984. 7. G u s t a f s s o n , L.L., H a r t v i g , P., Bergstrom, K., L i n d b e r g B., L u n d q v i s t , H., LQngstrom, B., Svard, H., Rane, A. & Tamsen, A.: S u b m i t t e d t o B r . J. A n a e s t h e s i a . f l u i d and t h e s p i n a l d u r a m a t e r . J. N e u r o l . Neurosurg. P s y c h i a t . 35: 9. O r r i s o n , W.W., E l d e v i k , O.P. & S a c h e t t , J.F.: L a t e r a l C 1 - C 2 p u n c t u r e f o r 8. M a r t i n s , A.N., W i l e y , J.K. & Myers, P.W.J.: Dynamics o f t h e c e r e b r o s p i n a l 468-473, 1972. c e r v i c a l myelography. P a t 111: h i s t o r i c a l , anatomic and t e c h n i c a l c o n s i d e r a t i o n . R a d i o l o g y 146: 401-408, 1983. 10. R i e s e l b a c h , R.E., d i C h i r o , G., F r e i r e i c h , E.J. & R a l l , D.P.: M e t h o t r e x a t e : d i s t r i b u t i o n i n c e r e b r o s p i n a l f l u i d a f t e r i n t r a v e n o u s , v e n t r i c u l a r and lumbar i n j e c t i o n . N. E n g l . J. Med. 267: 1273-1278, 1962. Address f o r r e p r i n t s : Ass p r o f e s s o r P e r H a r t v i g , Pharm.D., Ph.D. H o s p i t a l Pharmacy U n i v e r s i t y h o s p i t a l S-751 85 Uppsala, Sweden 213