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Brain Research, 555 (1991) 251-258 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116844Z BRES 16844
Spinal transection reduces both spinal antinociception and CNS concentration of systemically administered morphine in rats Claire A d v o k a t I a n d A n i l G u l a t i 2 1Department of Psychology, Louisiana State University, Baton Rouge, LA 70803 (U.S.A.) and 2Department of Pharmacodynamics, The University of Illinois at Chicago, Health Sciences Center, Chicago, IL (U.S.A.) (Accepted 5 March 1991) Key words: Spinal opiate antinociception; Spinal rat; Morphine radioimmunoassay
Within one day after spinal transection, the antinociceptive effect of systemically administered morphine on the spinal withdrawal reflex is significantly reduced. This observation has provided important empirical support for the present model of opiate-induced analgesia. One prediction from this model is that the antinociceptive effect of intrathecal (spinal) morphine injections should not be reduced by spinalization. When examined experimentally, this prediction was not supported; the antinociceptive effect of intrathecally administered morphine was significantly enhanced after acute spinalization. This result suggested an alternate hypothesis of morphine-induced analgesia. One prediction from this new hypothesis is that the decreased behavioral response to systemic morphine in spinal rats is due to a decrease in the spinal concentration of morphine produced by spinal transection. To test this prediction separate groups of intact rats and acute (one day) spinal rats, were assessed with the tail-flick (TF) procedure 60 min after subcutaneous injection of various doses of morphine (0.75, 1.5, 3.0, 4.5, 6.0 or 9.0 mg/kg) or at different time points (30, 60, 90, 150 or 240 min) after a single injection of 9.0 mg/kg. Immediately after behavioral testing, the rats were killed and brains, spinal cords and blood samples were collected and subsequently analyzed with a morphine radioimmunoassay. The results show that the concentration of morphine in the brain and spinal cords of acute spinal rats is significantly lower than that of intact rats, whereas morphine levels in the blood do not differ. These data suggest that the decreased antinociceptive effect of subcutaneous morphine in acute spinal rats is due to a decrease in the concentration of the opiate in the central nervous system. The data are consistent with a recently proposed model of morphine-induced analgesia and are discussed in the context of that hypothesis. INTRODUCTION Numerous studies have shown that the antinociceptive effect of systemically administered morphine, on the thermally elicited tail withdrawal reflex in rats, is significantly reduced by acute (1 day) spinal transection 2,7,1°,25. This decrease in spinal opiate action provides crucial empirical support for the most prevalent hypothesis concerning the neural substrate of morphine-induced analgesia 3. According to this generally accepted model, morphine produces analgesia by (1) a direct effect in the brain; (2) a direct effect in the spinal cord; and (3) a combined action at both of these sites. This combined action is thought to involve an increase in inhibitory control, exerted by descending supraspinal pathways on spinal reflex circuits. Presumably, it is the removal of this descending opiate-induced inhibition that is responsible for the decreased efficacy of systemic morphine after spinal transection. In the absence of descending inhibition, only the direct, local effect of morphine at the spinal cord is expressed. The result is a decreased antinociceptive effect of systemically administered morphine on spinal reflexes.
A substantial amount of evidence has provided support for this model. However, recent results from this laboratory led us to consider an alternate interpretation 1, 2,20,21. In those experiments, we assessed spinal antinociception in spinal rats, by using the intrathecal rather than the peripheral route of administration. We reasoned that the effect of intrathecal morphine might not be changed by spinal transection. That is, in both intact and spinal rats, the local effect of morphine on the spinal cord would not be accompanied by a corresponding influence of morphine at supraspinal sites. Therefore, the effect of intrathecal morphine in spinal animals might not differ from that observed in intact rats. However, this prediction was not supported; the antinociceptive effect of intrathecal morphine injections was profoundly increased in spinal vs intact rats. The ADso for intrathecal morphine was approximately 40x greater in intact rats than in spinal rats (5/~g vs 0.125/gg) 2°. This unexpected increase in the potency of intrathecal morphine in acute spinal rats suggested that the spinal action of morphine was influenced by the spinal transection. The data indicated that in the intact animal the
Correspondence: C. Advokat, Psychology Department, Louisiana State University, Baton Rouge, LA, 70803, U.S.A.
252 spinal effect of morphine was itself tonically inhibited by descending supraspinal input and that spinalization removed the inhibition and potentiated the antinociceptive response. Previous observations, from studies in intact rats, support this interpretation. It has been demonstrated that concurrent injection of morphine into the third cerebral ventricle26 or the periaqueductal gray 21 produces an analgesic response which is much greater than acute administration into each of these sites separately. This p h e n o m e n o n , termed the multiplicative or synergistic effect of morphine is particularly significant because it appears to be relevant to the normal physiological mechanisms that mediate opiate analgesia after systemic administration. In this situation, the ADs0 of intrathecal morphine is comparable to that observed in the spinal rat (approximately 0 . 1 5 - 0 . 2 0 / t g ) . This result suggests that both supraspinal morphine injections and spinal transection produce the same outcome, i.e. an increase in the potency of spinal morphine. It has been suggested that this occurs because both manipulations decrease descending inhibition of spinal opiate action 1. This interpretation offers a parsimonious explanation for the increased antinociceptive effects of intrathecal morphine in intact and spinal rats. However, one prediction from this model is that the effect of systemic morphine in spinal rats should be the same as that in intact rats, because in each case spinal opiate effects are expressed in the absence of descending inhibition. Why then is the antinociceptive response to systemic morphine reduced after spinal transection while the same response to intrathecal morphine is potentiated? O n e possibility is that the pharmacokinetics of systemic morphine is disturbed by the trauma of spinalization. As a result, the a m o u n t of morphine that reaches the spinal cord after subcutaneous injection is less than the a m o u n t that reaches the cord in intact animals. Consequently, the behavioral response is reduced. The present experiments were designed to examine this possibility. Separate groups of intact and spinal rats (transected one day earlier) were injected subcutaneously with different doses of morphine and assessed on the tail flick test. Immediately after the test the rats were killed and the brains, spinal cords and blood samples were collected and subsequently assayed for morphine. The results show that the concentration of morphine in the brains and spinal cords of acute spinal rats is significantly lower than that of intact rats, whereas morphine levels in the blood do not differ. These data suggest that the decreased antinociceptive effect of subcutaneous morphine in acute spinal rats is due to a decrease in the concentration of the opiate within the central nervous system. The results are consistent with the proposed
hypothesis, although a more thorough quantitative analysis of opiate pharmacokinetics may be required for a complete evaluation of the model. MATERIALS AND METHODS
Animals A total of 84 male, Sprague-Dawley-derived rats (Holtzman Laboratories, Madison, WI), weighing 215-330 g, were housed individually in suspended, stainless steel cages in a colony room maintained on a 12 h/12 h light/dark cycle, with dark onset at 17.00 h. Food and water were available ad libitum. Surgical procedures Spinal transections were conducted under ether anesthesia. A laminectomy was performed between T 6 and T9 and a 1-2 mm portion of the spinal cord was removed by excavation and replaced with gelfoam to reduce bleeding, after which the incision was sutured. Intact rats received no anesthesia or surgery. Immediately following the transection, spinalized rats were returned to their cages, which were placed on heating pads to maintain body temperature. The next morning, spinal rats were bathed and their urine was expressed manually, by the application of pressure on their bladders. Behavioral testing was conducted in the afternoon, approximately 20-24 h after the transection. Behavioral assessment Reactivity to a noxious stimulus was evaluated in intact and spinal rats with the tail-flick (TF) test. Noxious stimulation was produced by a beam of high intensity light focused on the tail. The response time was measured automatically and was defined as the interval between the onset of the heat stimulus and the abrupt flick of the tail. Each determination consisted of 3 trials; the mean score was taken as the response latency. In order to minimize tissue damage to the tail, animals not responding within 14 s were removed from the apparatus and assigned a response latency of 14 s. Nociception in intact rats was also assessed with the hot-plate (HP) test. A Plexiglas cylinder (28.5 cm in height x 20.5 cm in diameter) which restrained the rat was placed on top of a metal plate. The temperature of the plate was maintained at 54 + 1.0 °C by a heated water bath. Animals were placed within the cylinder and the latency until either the rat licked a hindpaw or jumped out of the cylinder was manually recorded. Animals not responding within 30 s were removed and assigned a response latency of 30 s. Each session consisted of a single trial. Drug administration For systemic administration, morphine sulphate (Penick Corporation, Lyndhurst, NJ) was dissolved in 0.9% saline and injected subcutaneously such that the appropriate dose (0.75-9.0 mg/kg) was administered in a volume of 1 ml/kg. Experimental procedures All rats were assessed on the TF apparatus prior to morphine injection, and intact rats were also assessed on the liP. In the first experiment, separate groups of intact and spinal rats were injected s.c. with one of the following doses of morphine: 0.75, 1.5, 3.0, 4.5, 6.0 or 9.0 mg/kg. At 60 min after the injection each rat was tested again on the TF, and intact rats were retested on the HE Immediately after this test, the rats were briefly anesthetized with ether until they lost consciousness at which point they were decapitated. Trunk blood samples were taken and the brains and spinal cords (below the point of transection) were rapidly removed, weighed, frozen on dry ice and stored at -70 °C for subsequent assay. The same procedure was followed in the second experiment, with the exception that all rats were injected with 9.0 mg/kg of morphine s.c. and then separate groups of intact rats were assessed on the two tests at 30, 60, 90, 150 or 240 rain later, while separate groups of
253 DOSE RESPONSE OF MORPHINE INDUCED ANALGESIA (60 rain intervol)
spinal rats were tested on the TF at 60, 150 or 240 min later.
Morphine radioimmunoassay Sample purification. The tissues were homogenized (Brinkman Polytron setting 5; 20 s) in 3 ml of 0.10 mM sodium dihydrogenphosphate (pH 2.1). Sample purification was carried out as described previously22 using Sep-Pak Cts cartridges (MilliporeWaters), which were conditioned with 5 ml of methanol, 3 ml of 10 mM sodium dihydrogenphosphate (pH 2.1) containing 10% acetonitrile and 5 ml water. Sample buffered with 3 ml of 500 mM ammonium sulfate (pH 9.3) was applied to the cartridge and after washing with 20 ml of 5 mM ammonium sulfate (pH 9.3), followed by 0.5 ml of water, morphine was eluted with 3 ml of 10 mM sodium dihydrogenphosphate (pH 2.1), containing 10% acetonitrile. The extract was then buffered with a further 3 ml of 500 mM ammonium sulfate and treated on a second cartridge in exactly the same way. This procedure resulted in a 92% recovery from the spiked samples. Estimation of morphine by radioimmunoassay. Morphine level was measured using [125I]morphine radioimmunoassay (RIA, COAT-A-COUNT, Diagnostics Products Corporation, LA) as described earlier~. The principle of the assay is based on 125Ilabelled morphine competing with morphine in the sample for antibody sites. Each RIA kit is equipped with standards having morphine values ranging from 0 to 250 ng/ml, and one vial of tracer [lzSI]morphine, which has high specific activity with total counts of approximately 80,000 cpm at iodination. Each kit also contains antibody-coated and uncoated polypropyline tubes. The antiserum has been found to be highly specific for unconjugated morphine with low cross-reactivity to morphine 3-glucuronide, morphine 6-glucuronide and codeine (o
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Opiate antinociception in spinal rats A c o m p a r i s o n of the baseline, pretest T F scores of all intact and spinal (n = 34) rats confirmed prior reports that T F latencies decrease significantly within one day after transection (intact m e a n = 4.5 + 0.09 s vs spinal m e a n = 2.6 + 0.12 s, P < 0.001). Fig. 3 shows the c o m p l e t e d o s e - r e s p o n s e function of spinal rats on the T F and the linear portion of the d o s e - r e s p o n s e function of intact rats (same as Fig. 1). A s seen in the figure, although there was a significant effect of dose ( P < 0.001), the function of spinal rats was shifted to the right
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with an ADs0 of 4.5 mg/kg (based on the values o b t a i n e d from the linear protion of the curve). Fig. 4 shows the time course of o p i a t e antinociception in spinal rats as well as the corresponding scores of intact rats (taken from Fig. 2). T h e r e was a significant decrease in antinociception in spinal rats as a function of time after s.c. injection (P < 0.001). A two-way analysis of variance was p e r f o r m e d on the d a t a of intact and spinal rats at the last two intervals of 150 and 240 min, i.e. when the scores were below the cut-off values. The result showed a significant difference b e t w e e n the two conditions ( P < 0.01) and a significant effect of time interval ( P < 0.01) with no interaction ( P = 0.66). To our k n o w l e d g e this is the first d e m o n s t r a t i o n of the time course of opiate antinociception in spinal rats. T h e results indicate that the relative decrease in o p i a t e antinociception in spinal vs intact rats is m a i n t a i n e d for several hours after an acute s.c. injection.
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Figs. 5 - 1 0 summarize the results of the m o r p h i n e radioimmunoassays in intact and spinal rats. D u e to technical difficulties it was not possible to obtain all 3 values for each rat. Statistical analysis of the d a t a p r e s e n t e d in Fig. 5 confirmed that t h e r e was a significant increase in m o r p h i n e concentration in the b l o o d of intact (n --- 30) and spinal (n -- 24) rats with increasing morphine doses ( P < 0.001). H o w e v e r , there was no difference in concentration and no interaction b e t w e e n the two e x p e r i m e n t a l conditions. Fig. 6 shows that t h e r e was also a significant increase in m o r p h i n e concentration as a function of dose, in the
255 MORPHINE CONCENTRATION IN THE BRAIN OF INTACT AND SPINAL RATS (60 rain interv01)
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brains of intact (n = 27) and spinal (n = 21) rats (P < 0.001). In this case, however, there was also a significant difference between the two conditions (P -- 0.0004) and a borderline significant interaction (P = 0.0455). This result indicates that morphine concentration in the brains of spinal rats is significantly lower than the concentration in the brains of intact rats, and suggests that this difference is larger at the higher doses. Fig. 7 shows that the same result was obtained in the spinal cord. There was a significant increase in spinal morphine concentration as a function of dose (intact n = 29, spinal n = 24; P < 0.001) and a significant difference between the two experimental conditions (P = 0.0011). In this case, the interaction did not reach significance.
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256
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varied between 29 and 35%. There was no significant relationship between the dose of morphine and the percent of drug in the brain nor was there a difference across doses between intact and spinal rats. This result indicates that at 60 min after a s.c. injection the amount of morphine in the brain is approximately one third of the total amount in the CNS, and the amount in the spinal cord is approximately two-thirds. Furthermore, the data show that this relationship is maintained for at least one day after spinal transection. Fig. 8 shows the concentration of morphine in the blood of intact (n = 25) and spinal (n = 14) rats at several time points after s.c. injection of 9.0 mg/kg. A two-way A N O V A performed on the 3 common time points indicated that there was a significant decrease over time (P < 0.001), with no difference between intact and spinal groups and no interaction. Figs. 9 and 10 show the morphine concentration in the brains and spinal cords, respectively, of the same groups of rats, as a function of time after morphine administration. Analyses of these data showed a significant effect of time (P < 0.001 in each case), a significant difference between the two experimental conditions (P = 0.0006 for brain and P = 0.0058 for spinal cord) and a significant interaction (P < 0.001 for brain and P = 0.0110 for spinal cord). These data suggest that the difference in morphine concentration in the CNS of spinal vs intact rats is maintained for several hours after s.c. drug administration. However, the fact that there was also a significant interaction suggests that morphine leaves the central nervous system of spinal rats more slowly than the CNS of intact rats.
DISCUSSION The present studies were designed to evaluate a hypothesis derived from two, apparently contradictory, behavioral observations regarding opiate antinociception in spinal rats. The first observation is that the antinociceptive effect of systemic morphine on spinal reflexes is reduced by spinal transection. This phenomenon has generally been interpreted to mean that, under normal conditions, i.e. in intact animals, the spinal action of morphine is potentiated by a concomitant action of morphine in the brain. That is, systemic morphine is believed to produce analgesia not only by a direct effect on the brain and spinal cord, but also by strengthening supraspinal inhibition onto spinal reflex circuits. According to this view, spinal opiate antinociception is reduced after spinal transection because this descending inhibition is eliminated. If this interpretation if correct, it would be expected that the antinociceptive effect of intrathecal morphine would be unchanged by spinal transection (see Introduction for this rationale). When this question was experimentally addressed, this prediction was not supported. The antinociceptive effect of spinal morphine was greatly potentiated, relative to the effect in intact rats 2°. This seemingly contradictory finding led to a reinterpretation of the role of descending inhibition in opiate analgesia 1. It was hypothesized that, in the intact animal, descending input inhibited the local effect of morphine on the spinal cord. When this inhibition was eliminated by spinal transection, the potency of intrathecal morphine was increased. However, this alternate hypothesis raised the question: why is the spinal effect of systemic morphine reduced by acute spinalization? One possible explanation is that the amount of morphine that reaches the spinal cord might also be reduced by the trauma of a spinal transection. A decrease in the concentration of morphine in the spinal cord might account for the decreased behavioral response. The present study was designed to answer that question. Both behavioral antinociception and morphine concentration in the spinal cord of intact and spinal rats were assessed after several doses and time intervals following subcutaneously administered morphine. The behavioral effect of s.c. morphine in intact and spinal rats in this study is consistent with previously reported results. Opiate antinociception was significantly increased as a function of dose and significantly decreased as a function of the post-injection interval, in both intact and spinal rats. In each case, however, the response of spinal rats was significantly less than that of intact rats. In this study, the ADs0 for spinal rats (4.5 mg/kg) was
257 3x greater than the ADs0 for intact rats (1.5 mg/kg). This difference is larger than that obtained in a previous study, which reported ADso'S of 1.6 and 2.25 mg/kg for intact and spinal rats, respectively2. However, in that previous study, animals were tested 30 rain after each dose, whereas in the present study the tests were administered 60 min after the injections. It is possible that this procedural change was responsible for the fact that the ADso value of the spinal rats reported here is larger than that obtained previously. This is implied by the results of the time course study, which suggests that the behavioral response of spinal rats declines more rapidly than the response of intact rats. The data are, therefore, consistent with all previously published reports showing a reduced antinociceptive effect of systemic morphine in acute spinal rats. There are surprisingly few reports in the literature regarding morphine concentration in the blood or the CNS of rats, after systemic administration, particularly with respect to the spinal cord. Those data that are available were obtained with a variety of methods, both in terms of the route of administration (subcutaneous, intravenous or intraperitoneal injections, or infusions and pellet implantation) and the assay technique (gas-liquid chromatography, spectroflourometry, high performance liquid chromatography or radioimmunoassay) 4'6's'9,1113,16-18,24. In many cases only one dose of morphine was examined; in others a chronic injection or infusion schedule was used. Consequently, there is a wide variation of reported morphine concentrations for both blood and brain, as a function of dose or post-injection interval. Therefore, it is not unexpected that our values generally fall in the middle of this broad range. Our data are consistent with previous reports in showing that morphine concentration in all tissues is positively correlated with dose and negatively correlated with post-injection interval. The peak blood value in this study occurred at 30 min, which was the earliest time point measured. Other studies, particularly those using the intravenous route 6,17, obtained peak levels within 5-15 min. Because we did not take samples at earlier time points it is possible that peak blood levels might have occurred in less than 30 min. However, our data do show that, in intact rats, blood levels decline between 30 and 60 min, whereas morphine concentrations in the brain and spinal cord increase during that time, and then subsequently decline from 60 to 240 min. Unfortunately, we do not know whether spinal rats also show this difference in time course between peak morphine concentrations in the blood and CNS, because the earliest time point assayed in spinal rats was 60 min. These results clearly show, first, that at the dose and time points examined in this study blood morphine
concentration is not altered by acute spinal transection. These data suggest that spinalization does not disrupt the absorption of morphine into the circulation after subcutaneous injection. Second, the present results show that after subcutaneous injection morphine is not equally distributed between the brain and the spinal cord. At 60 min after an acute injection, approximately one-third of the total amount in the CNS is in the brain, and two-thirds are in the spinal cord. This differential distribution was consistent across all doses. Furthermore, the present data show that this relationship was not disrupted by spinal transection. Rats that were spinally transected 24 h previously had the same proportion of morphine in the brain and spinal cord as intact rats. However, although the distribution of morphine within the CNS was the same in intact vs spinal rats, the present data also show that the absolute amount of morphine at both sites in the CNS is significantly reduced by spinal transection. There was a significant decrease in morphine levels within the CNS across the doses and the time points examined in this study. These data are consistent with the experimental hypothesis that the decrease in behavioral opiate antinociception in acute spinal rats, after subcutaneous administration, is due to a decrease in spinal morphine concentration. The fact that morphine levels in both the brain and the spinal cord were reduced by spinalization whereas blood levels were not, suggests that the mechanism responsible involves a disturbance in (1) the integrity of the bloodbrain barrier; (2) blood flow within the CNS; or (3) both. The effects of spinal cord transection on the blood-spinal cord barrier have been examined and described at both the light and electron microscopic levels 15. These studies have found that there is disruption of the vasculature along the cord within 24 h after spinalization, and that it is more pronounced at regions that are 1.0-2.0 cm distal to the transection than at proximal sites. This demonstration of vascular damage suggests that spinalization might promote the entry of morphine into the CNS, or at least into the spinal cord. Such a prediction is not supported by the present data, since CNS morphine levels were reduced, rather than increased, after the transection. This outcome implies that the decrease in morphine levels is not simply a consequence of the trauma associated with spinalization, although the relationship between vascular damage and transport of morphine into the spinal cord remains to be empirically determined (e.g. see ref. 14). However, the results of a recent study by Sinclair et al. 19 also suggest that the decrease in CNS morphine levels may not simply be due to the physical trauma of spinalization. In that experiment, the TF reflex was elicited in intact rats after either systemic or intrathecal
258 m o r p h i n e injections. In each of these two conditions, the latency was m e a s u r e d before and after the spinal cord was reversibly cooled to 2 °C. These authors r e p o r t e d that the reversible cold block of the spinal cord decreased the antinociceptive effect of systemic morphine on the TF, but increased the antinociceptive effect of intrathecal morphine. This is the same response pattern seen in spinally transected rats. Presumably, a cold-block of the spinal cord does not d a m a g e spinal vasculature. On the o t h e r hand, this t r e a t m e n t might reduce blood flow, not only at the level of the spinal cord but throughout the CNS. A l t h o u g h it has been r e p o r t e d that spinal cord b l o o d flow is not i m p a i r e d after spinal transection, these measures were t a k e n only within the first 15 min after spinalization 23.
REFERENCES 1 Advokat, C., The role of descending inhibition in morphineinduced analgesia, Trends Pharmacol. Sci., 9 (1988) 330-334. 2 Advokat, C. and Burton, P., Antinociceptive effect of systemic and intrathecal morphine in spinally transected rats, Eur. J. Pharmacol., 139 (1987) 335-343. 3 Basbaum, A.I. and Fields, H.L., Endogenous pain control systems: bralnstem spinal pathways and endorphin circuitry, Annu. Rev. Neurosci., 7 (1984) 309-338. 4 Berkowitz, B.A., Cerreta, K.V. and Spector, S., The influence of physiologic and pharmacologic factors on the disposition of morphine as determined by radioimmunoassay, J. Pharmacol. Exp. Ther., 191 (1974) 527-534. 5 Bhat, R., Chaff, G., Gulati, A., Aidana, O., Velamati, R. and Bhargava, H.N., Pharmacokinetics of a single dose of morphine in preterm infants during the first week of life, J. Pediatr., 117 (1990) 477-481. 6 Bolander, H., Kourtopoulos, H., Lundberg, S. and Persson, S.-A., Morphine concentrations in serum, brain and cerebrospinal fluid in the rat after intravenous administration of a single dose, J. Pharm. Pharmacol., 35 (1983) 656-659. 7 Bonnycastle, D.D., Cook, L. and Ipsen, J., The action of some analgesic drugs in intact and chronic spinal rats, Acta Pharmacol. Toxicol., 9 (1953) 332-336. 8 Dahlstrom, B.E., Paalzow, L.K., Segre, G. and Agren, A.J., Relation between morphine pharmacokinetics and analgesia, J. Pharmacokin. Biopharm., 6 (1978) 41-53. 9 Hipps, P.P., Eveland, M.R., Meyer, E.R., Sherman, W.R. and Cicero, T.J., Mass fragmentography of morphine: relationship between brain levels and analgesic activity, J. Pharmacol. Exp. Ther., 196 (1976) 642-648. 10 Irwin, S., Houde, R.W., Bennet, D.R., Hendershot, L.C. and Seevers, M.H., The effects of morphine, methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation, J. Pharmacol. Exp. Ther. , 101 (1951) 132-143. 11 Johannesson, T. and Schou, J., Analgesic activity and brain concentration of morphine in tolerant and nontolerant rats given morphine alone or with neostigmine, Acta Pharmacol. Toxicol., 20 (1963) 213-221. 12 Johannesson, T. and Woods, L.A., Analgesic action and brain and plasma levels of morphine and codeine in morphine tolerant, codeine tolerant and non-tolerant rats, Acta Pharmacol. Toxicol., 21 (1964) 381-396. 13 Lesher, G.A. and Spratto, G.R., Brain and plasma concentrations of morphine during the development of physical dependence and tolerance, J. Pharm. Pharmacol., 28 (1976) 843-844.
Regardless of the mechanism involved, the fact that both spinalization and spinal cold block p r o d u c e d similar responses to both systemic and spinal morphine, is consistent with the present m o d e l , which proposes that in each of these situations, the effect of intrathecal morphine is enhanced because descending inhibitory control is r e m o v e d , and the effect of systemic morphine is reduced, because the m o v e m e n t of m o r p h i n e from the blood into the CNS is impaired. A d d i t i o n a l research will be required to u n d e r s t a n d the mechanism responsible for this impairment. Acknowledgements. This work was supported by USPHS Grant DA 02845. We thank J. Magoun, S. McCann and C. Mclnnis for technical assistance in performing the spinal transections and collecting the tissue samples.
14 Milne, R.J., Coddington, J.M. and Gamble, G.D., Quaternary naloxone blocks morphine analgesia in spinal but not intact rats, Neurosci. Lett., 114 (1990) 259-264. 15 Noble, L.J. and Wrathall, J.R., The blood spinal-cord barrier after injury: pattern of vascular events proximal and distal to a transection in the rat, Brain Research, 424 (1987) 177-188. 16 Patrick, G.A., Dewey, W.L., Huger, EP., Dares, E.D. and Harris, L., Disposition of morphine in chronically infused rats: relationship to antinociception and tolerance, J. Pharmacol. Exp. Ther., 205 (1978) 556-562. 17 Plomp, G.J.J., Maes, R.A.A. and Van Ree, J.M., Disposition of morphine in rat brain: relationship to biological activity, J. Pharmacol. Exp. Ther., 217 (1981) 181-188. 18 Raffa, R.B., Porreca, F., Cowan, A. and Tallarida, R.J., Extraction and measurement of morphine: correlation of brain level and s.c. dose in drug-naive and morphine-tolerant rats, Life Sci., 31 (1982) 2299-2302. 19 Sinclair, J.G., Main, C.D. and Lo, C.F., Spinal vs supraspinal actions of morphine on the rat tall-flick reflex, Pain, 33 (1988) 357-362. 20 Siuciak, J.A. and Advokat, C., Antinociceptive effect of intrathecal morphine in tolerant and non-tolerant spinal rats, Pharmacol. Biochem. Behav., 34 (1989) 445-452. 21 Siuciak, J.A. and Advokat, C., The synergistic effect of concurrent spinal and supraspinal opiate agonisms is reduced by both nociceptive and morphine pretreatment, Pharmacol. Biochem. Behav., 34 (1989) 265-273. 22 Svensson, J.O., Rane, A., Sawe, J. and Sjoqvist, E, Determination of morphine, morphine 3-giucuronide and (tentatively) morphine 6-glucuronide in plasma and urine using ion-pair high-performance liquid chromatography, J. Chromatogr., 230 (1982) 427-432. 23 Turner, J. and Heavner, J.E., Influence of spinal cord transection on spinal cord blood flow in rats, Gen. Pharmacol., 9 (1978) 463-465. 24 Vetulani, J., Melzacka, M., Adamus, A. and Danek, L., Changes in morphine pharmacokinetics in nervous and peripheral tissues following different schedules of administration, Arch. Int. Pharmacodyn., 265 (1983) 180-191. 25 Wood, P.L., Rackham, A. and Richard, J., Spinal analgesia: comparison of the /z agonist morphine and the K agonist ethylketazocine, Life Sci., 28 (1981) 2119-2125. 26 Yeung, J.C. and Rudy, T.A., Multiplicative interaction between narcotic agonisms expressed at spinal and supraspinal sites of antinociceptive action as revealed by concurrent intrathecal and intracerebroventricular injections of morphine, J. Pharmacol. Exp. Ther., 215 (1980) 633-642.
Brain Research, 555 (1991) 251-258 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116844Z BRES 16844
Spinal transection reduces both spinal antinociception and CNS concentration of systemically administered morphine in rats Claire A d v o k a t I a n d A n i l G u l a t i 2 1Department of Psychology, Louisiana State University, Baton Rouge, LA 70803 (U.S.A.) and 2Department of Pharmacodynamics, The University of Illinois at Chicago, Health Sciences Center, Chicago, IL (U.S.A.) (Accepted 5 March 1991) Key words: Spinal opiate antinociception; Spinal rat; Morphine radioimmunoassay
Within one day after spinal transection, the antinociceptive effect of systemically administered morphine on the spinal withdrawal reflex is significantly reduced. This observation has provided important empirical support for the present model of opiate-induced analgesia. One prediction from this model is that the antinociceptive effect of intrathecal (spinal) morphine injections should not be reduced by spinalization. When examined experimentally, this prediction was not supported; the antinociceptive effect of intrathecally administered morphine was significantly enhanced after acute spinalization. This result suggested an alternate hypothesis of morphine-induced analgesia. One prediction from this new hypothesis is that the decreased behavioral response to systemic morphine in spinal rats is due to a decrease in the spinal concentration of morphine produced by spinal transection. To test this prediction separate groups of intact rats and acute (one day) spinal rats, were assessed with the tail-flick (TF) procedure 60 min after subcutaneous injection of various doses of morphine (0.75, 1.5, 3.0, 4.5, 6.0 or 9.0 mg/kg) or at different time points (30, 60, 90, 150 or 240 min) after a single injection of 9.0 mg/kg. Immediately after behavioral testing, the rats were killed and brains, spinal cords and blood samples were collected and subsequently analyzed with a morphine radioimmunoassay. The results show that the concentration of morphine in the brain and spinal cords of acute spinal rats is significantly lower than that of intact rats, whereas morphine levels in the blood do not differ. These data suggest that the decreased antinociceptive effect of subcutaneous morphine in acute spinal rats is due to a decrease in the concentration of the opiate in the central nervous system. The data are consistent with a recently proposed model of morphine-induced analgesia and are discussed in the context of that hypothesis. INTRODUCTION Numerous studies have shown that the antinociceptive effect of systemically administered morphine, on the thermally elicited tail withdrawal reflex in rats, is significantly reduced by acute (1 day) spinal transection 2,7,1°,25. This decrease in spinal opiate action provides crucial empirical support for the most prevalent hypothesis concerning the neural substrate of morphine-induced analgesia 3. According to this generally accepted model, morphine produces analgesia by (1) a direct effect in the brain; (2) a direct effect in the spinal cord; and (3) a combined action at both of these sites. This combined action is thought to involve an increase in inhibitory control, exerted by descending supraspinal pathways on spinal reflex circuits. Presumably, it is the removal of this descending opiate-induced inhibition that is responsible for the decreased efficacy of systemic morphine after spinal transection. In the absence of descending inhibition, only the direct, local effect of morphine at the spinal cord is expressed. The result is a decreased antinociceptive effect of systemically administered morphine on spinal reflexes.
A substantial amount of evidence has provided support for this model. However, recent results from this laboratory led us to consider an alternate interpretation 1, 2,20,21. In those experiments, we assessed spinal antinociception in spinal rats, by using the intrathecal rather than the peripheral route of administration. We reasoned that the effect of intrathecal morphine might not be changed by spinal transection. That is, in both intact and spinal rats, the local effect of morphine on the spinal cord would not be accompanied by a corresponding influence of morphine at supraspinal sites. Therefore, the effect of intrathecal morphine in spinal animals might not differ from that observed in intact rats. However, this prediction was not supported; the antinociceptive effect of intrathecal morphine injections was profoundly increased in spinal vs intact rats. The ADso for intrathecal morphine was approximately 40x greater in intact rats than in spinal rats (5/~g vs 0.125/gg) 2°. This unexpected increase in the potency of intrathecal morphine in acute spinal rats suggested that the spinal action of morphine was influenced by the spinal transection. The data indicated that in the intact animal the
Correspondence: C. Advokat, Psychology Department, Louisiana State University, Baton Rouge, LA, 70803, U.S.A.
252 spinal effect of morphine was itself tonically inhibited by descending supraspinal input and that spinalization removed the inhibition and potentiated the antinociceptive response. Previous observations, from studies in intact rats, support this interpretation. It has been demonstrated that concurrent injection of morphine into the third cerebral ventricle26 or the periaqueductal gray 21 produces an analgesic response which is much greater than acute administration into each of these sites separately. This p h e n o m e n o n , termed the multiplicative or synergistic effect of morphine is particularly significant because it appears to be relevant to the normal physiological mechanisms that mediate opiate analgesia after systemic administration. In this situation, the ADs0 of intrathecal morphine is comparable to that observed in the spinal rat (approximately 0 . 1 5 - 0 . 2 0 / t g ) . This result suggests that both supraspinal morphine injections and spinal transection produce the same outcome, i.e. an increase in the potency of spinal morphine. It has been suggested that this occurs because both manipulations decrease descending inhibition of spinal opiate action 1. This interpretation offers a parsimonious explanation for the increased antinociceptive effects of intrathecal morphine in intact and spinal rats. However, one prediction from this model is that the effect of systemic morphine in spinal rats should be the same as that in intact rats, because in each case spinal opiate effects are expressed in the absence of descending inhibition. Why then is the antinociceptive response to systemic morphine reduced after spinal transection while the same response to intrathecal morphine is potentiated? O n e possibility is that the pharmacokinetics of systemic morphine is disturbed by the trauma of spinalization. As a result, the a m o u n t of morphine that reaches the spinal cord after subcutaneous injection is less than the a m o u n t that reaches the cord in intact animals. Consequently, the behavioral response is reduced. The present experiments were designed to examine this possibility. Separate groups of intact and spinal rats (transected one day earlier) were injected subcutaneously with different doses of morphine and assessed on the tail flick test. Immediately after the test the rats were killed and the brains, spinal cords and blood samples were collected and subsequently assayed for morphine. The results show that the concentration of morphine in the brains and spinal cords of acute spinal rats is significantly lower than that of intact rats, whereas morphine levels in the blood do not differ. These data suggest that the decreased antinociceptive effect of subcutaneous morphine in acute spinal rats is due to a decrease in the concentration of the opiate within the central nervous system. The results are consistent with the proposed
hypothesis, although a more thorough quantitative analysis of opiate pharmacokinetics may be required for a complete evaluation of the model. MATERIALS AND METHODS
Animals A total of 84 male, Sprague-Dawley-derived rats (Holtzman Laboratories, Madison, WI), weighing 215-330 g, were housed individually in suspended, stainless steel cages in a colony room maintained on a 12 h/12 h light/dark cycle, with dark onset at 17.00 h. Food and water were available ad libitum. Surgical procedures Spinal transections were conducted under ether anesthesia. A laminectomy was performed between T 6 and T9 and a 1-2 mm portion of the spinal cord was removed by excavation and replaced with gelfoam to reduce bleeding, after which the incision was sutured. Intact rats received no anesthesia or surgery. Immediately following the transection, spinalized rats were returned to their cages, which were placed on heating pads to maintain body temperature. The next morning, spinal rats were bathed and their urine was expressed manually, by the application of pressure on their bladders. Behavioral testing was conducted in the afternoon, approximately 20-24 h after the transection. Behavioral assessment Reactivity to a noxious stimulus was evaluated in intact and spinal rats with the tail-flick (TF) test. Noxious stimulation was produced by a beam of high intensity light focused on the tail. The response time was measured automatically and was defined as the interval between the onset of the heat stimulus and the abrupt flick of the tail. Each determination consisted of 3 trials; the mean score was taken as the response latency. In order to minimize tissue damage to the tail, animals not responding within 14 s were removed from the apparatus and assigned a response latency of 14 s. Nociception in intact rats was also assessed with the hot-plate (HP) test. A Plexiglas cylinder (28.5 cm in height x 20.5 cm in diameter) which restrained the rat was placed on top of a metal plate. The temperature of the plate was maintained at 54 + 1.0 °C by a heated water bath. Animals were placed within the cylinder and the latency until either the rat licked a hindpaw or jumped out of the cylinder was manually recorded. Animals not responding within 30 s were removed and assigned a response latency of 30 s. Each session consisted of a single trial. Drug administration For systemic administration, morphine sulphate (Penick Corporation, Lyndhurst, NJ) was dissolved in 0.9% saline and injected subcutaneously such that the appropriate dose (0.75-9.0 mg/kg) was administered in a volume of 1 ml/kg. Experimental procedures All rats were assessed on the TF apparatus prior to morphine injection, and intact rats were also assessed on the liP. In the first experiment, separate groups of intact and spinal rats were injected s.c. with one of the following doses of morphine: 0.75, 1.5, 3.0, 4.5, 6.0 or 9.0 mg/kg. At 60 min after the injection each rat was tested again on the TF, and intact rats were retested on the HE Immediately after this test, the rats were briefly anesthetized with ether until they lost consciousness at which point they were decapitated. Trunk blood samples were taken and the brains and spinal cords (below the point of transection) were rapidly removed, weighed, frozen on dry ice and stored at -70 °C for subsequent assay. The same procedure was followed in the second experiment, with the exception that all rats were injected with 9.0 mg/kg of morphine s.c. and then separate groups of intact rats were assessed on the two tests at 30, 60, 90, 150 or 240 rain later, while separate groups of
253 DOSE RESPONSE OF MORPHINE INDUCED ANALGESIA (60 rain intervol)
spinal rats were tested on the TF at 60, 150 or 240 min later.
Morphine radioimmunoassay Sample purification. The tissues were homogenized (Brinkman Polytron setting 5; 20 s) in 3 ml of 0.10 mM sodium dihydrogenphosphate (pH 2.1). Sample purification was carried out as described previously22 using Sep-Pak Cts cartridges (MilliporeWaters), which were conditioned with 5 ml of methanol, 3 ml of 10 mM sodium dihydrogenphosphate (pH 2.1) containing 10% acetonitrile and 5 ml water. Sample buffered with 3 ml of 500 mM ammonium sulfate (pH 9.3) was applied to the cartridge and after washing with 20 ml of 5 mM ammonium sulfate (pH 9.3), followed by 0.5 ml of water, morphine was eluted with 3 ml of 10 mM sodium dihydrogenphosphate (pH 2.1), containing 10% acetonitrile. The extract was then buffered with a further 3 ml of 500 mM ammonium sulfate and treated on a second cartridge in exactly the same way. This procedure resulted in a 92% recovery from the spiked samples. Estimation of morphine by radioimmunoassay. Morphine level was measured using [125I]morphine radioimmunoassay (RIA, COAT-A-COUNT, Diagnostics Products Corporation, LA) as described earlier~. The principle of the assay is based on 125Ilabelled morphine competing with morphine in the sample for antibody sites. Each RIA kit is equipped with standards having morphine values ranging from 0 to 250 ng/ml, and one vial of tracer [lzSI]morphine, which has high specific activity with total counts of approximately 80,000 cpm at iodination. Each kit also contains antibody-coated and uncoated polypropyline tubes. The antiserum has been found to be highly specific for unconjugated morphine with low cross-reactivity to morphine 3-glucuronide, morphine 6-glucuronide and codeine (o
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Opiate antinociception in spinal rats A c o m p a r i s o n of the baseline, pretest T F scores of all intact and spinal (n = 34) rats confirmed prior reports that T F latencies decrease significantly within one day after transection (intact m e a n = 4.5 + 0.09 s vs spinal m e a n = 2.6 + 0.12 s, P < 0.001). Fig. 3 shows the c o m p l e t e d o s e - r e s p o n s e function of spinal rats on the T F and the linear portion of the d o s e - r e s p o n s e function of intact rats (same as Fig. 1). A s seen in the figure, although there was a significant effect of dose ( P < 0.001), the function of spinal rats was shifted to the right
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with an ADs0 of 4.5 mg/kg (based on the values o b t a i n e d from the linear protion of the curve). Fig. 4 shows the time course of o p i a t e antinociception in spinal rats as well as the corresponding scores of intact rats (taken from Fig. 2). T h e r e was a significant decrease in antinociception in spinal rats as a function of time after s.c. injection (P < 0.001). A two-way analysis of variance was p e r f o r m e d on the d a t a of intact and spinal rats at the last two intervals of 150 and 240 min, i.e. when the scores were below the cut-off values. The result showed a significant difference b e t w e e n the two conditions ( P < 0.01) and a significant effect of time interval ( P < 0.01) with no interaction ( P = 0.66). To our k n o w l e d g e this is the first d e m o n s t r a t i o n of the time course of opiate antinociception in spinal rats. T h e results indicate that the relative decrease in o p i a t e antinociception in spinal vs intact rats is m a i n t a i n e d for several hours after an acute s.c. injection.
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Figs. 5 - 1 0 summarize the results of the m o r p h i n e radioimmunoassays in intact and spinal rats. D u e to technical difficulties it was not possible to obtain all 3 values for each rat. Statistical analysis of the d a t a p r e s e n t e d in Fig. 5 confirmed that t h e r e was a significant increase in m o r p h i n e concentration in the b l o o d of intact (n --- 30) and spinal (n -- 24) rats with increasing morphine doses ( P < 0.001). H o w e v e r , there was no difference in concentration and no interaction b e t w e e n the two e x p e r i m e n t a l conditions. Fig. 6 shows that t h e r e was also a significant increase in m o r p h i n e concentration as a function of dose, in the
255 MORPHINE CONCENTRATION IN THE BRAIN OF INTACT AND SPINAL RATS (60 rain interv01)
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In addition to these results, comparison of Figs. 6 and 7 suggests an additional finding in regard to morphine concentration in the central nervous system. At each dose, the amount of morphine in the spinal cord appeared to be greater than in the brain, for both intact and spinal rats. To quantifiy this observation, the percent of the total drug concentration in the brain was determined for each rat. These values were obtained by calculating the ratio of brain concentration/spinal cord + brain concentration x 100 (where both values were available). For intact rats (n = 26) the mean percent of morphine in the brain varied between 26 and 40% for the various doses. For spinal rats (n = 21) this value
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brains of intact (n = 27) and spinal (n = 21) rats (P < 0.001). In this case, however, there was also a significant difference between the two conditions (P -- 0.0004) and a borderline significant interaction (P = 0.0455). This result indicates that morphine concentration in the brains of spinal rats is significantly lower than the concentration in the brains of intact rats, and suggests that this difference is larger at the higher doses. Fig. 7 shows that the same result was obtained in the spinal cord. There was a significant increase in spinal morphine concentration as a function of dose (intact n = 29, spinal n = 24; P < 0.001) and a significant difference between the two experimental conditions (P = 0.0011). In this case, the interaction did not reach significance.
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POST INJECTION TIME (rain)
Fig. 10. Morphine concentration in the spinal cord of intact (filled circles, n = 25) and acute spinal rats (filled triangles, n = 14) at 30, 60, 90, 150 and 240 min after s.c. injection of 9.0 mg/kg. Each point represents the mean ng/g of tissue of those groups whose antinociceptive response is shown in Figs. 2 and 4.
varied between 29 and 35%. There was no significant relationship between the dose of morphine and the percent of drug in the brain nor was there a difference across doses between intact and spinal rats. This result indicates that at 60 min after a s.c. injection the amount of morphine in the brain is approximately one third of the total amount in the CNS, and the amount in the spinal cord is approximately two-thirds. Furthermore, the data show that this relationship is maintained for at least one day after spinal transection. Fig. 8 shows the concentration of morphine in the blood of intact (n = 25) and spinal (n = 14) rats at several time points after s.c. injection of 9.0 mg/kg. A two-way A N O V A performed on the 3 common time points indicated that there was a significant decrease over time (P < 0.001), with no difference between intact and spinal groups and no interaction. Figs. 9 and 10 show the morphine concentration in the brains and spinal cords, respectively, of the same groups of rats, as a function of time after morphine administration. Analyses of these data showed a significant effect of time (P < 0.001 in each case), a significant difference between the two experimental conditions (P = 0.0006 for brain and P = 0.0058 for spinal cord) and a significant interaction (P < 0.001 for brain and P = 0.0110 for spinal cord). These data suggest that the difference in morphine concentration in the CNS of spinal vs intact rats is maintained for several hours after s.c. drug administration. However, the fact that there was also a significant interaction suggests that morphine leaves the central nervous system of spinal rats more slowly than the CNS of intact rats.
DISCUSSION The present studies were designed to evaluate a hypothesis derived from two, apparently contradictory, behavioral observations regarding opiate antinociception in spinal rats. The first observation is that the antinociceptive effect of systemic morphine on spinal reflexes is reduced by spinal transection. This phenomenon has generally been interpreted to mean that, under normal conditions, i.e. in intact animals, the spinal action of morphine is potentiated by a concomitant action of morphine in the brain. That is, systemic morphine is believed to produce analgesia not only by a direct effect on the brain and spinal cord, but also by strengthening supraspinal inhibition onto spinal reflex circuits. According to this view, spinal opiate antinociception is reduced after spinal transection because this descending inhibition is eliminated. If this interpretation if correct, it would be expected that the antinociceptive effect of intrathecal morphine would be unchanged by spinal transection (see Introduction for this rationale). When this question was experimentally addressed, this prediction was not supported. The antinociceptive effect of spinal morphine was greatly potentiated, relative to the effect in intact rats 2°. This seemingly contradictory finding led to a reinterpretation of the role of descending inhibition in opiate analgesia 1. It was hypothesized that, in the intact animal, descending input inhibited the local effect of morphine on the spinal cord. When this inhibition was eliminated by spinal transection, the potency of intrathecal morphine was increased. However, this alternate hypothesis raised the question: why is the spinal effect of systemic morphine reduced by acute spinalization? One possible explanation is that the amount of morphine that reaches the spinal cord might also be reduced by the trauma of a spinal transection. A decrease in the concentration of morphine in the spinal cord might account for the decreased behavioral response. The present study was designed to answer that question. Both behavioral antinociception and morphine concentration in the spinal cord of intact and spinal rats were assessed after several doses and time intervals following subcutaneously administered morphine. The behavioral effect of s.c. morphine in intact and spinal rats in this study is consistent with previously reported results. Opiate antinociception was significantly increased as a function of dose and significantly decreased as a function of the post-injection interval, in both intact and spinal rats. In each case, however, the response of spinal rats was significantly less than that of intact rats. In this study, the ADs0 for spinal rats (4.5 mg/kg) was
257 3x greater than the ADs0 for intact rats (1.5 mg/kg). This difference is larger than that obtained in a previous study, which reported ADso'S of 1.6 and 2.25 mg/kg for intact and spinal rats, respectively2. However, in that previous study, animals were tested 30 rain after each dose, whereas in the present study the tests were administered 60 min after the injections. It is possible that this procedural change was responsible for the fact that the ADso value of the spinal rats reported here is larger than that obtained previously. This is implied by the results of the time course study, which suggests that the behavioral response of spinal rats declines more rapidly than the response of intact rats. The data are, therefore, consistent with all previously published reports showing a reduced antinociceptive effect of systemic morphine in acute spinal rats. There are surprisingly few reports in the literature regarding morphine concentration in the blood or the CNS of rats, after systemic administration, particularly with respect to the spinal cord. Those data that are available were obtained with a variety of methods, both in terms of the route of administration (subcutaneous, intravenous or intraperitoneal injections, or infusions and pellet implantation) and the assay technique (gas-liquid chromatography, spectroflourometry, high performance liquid chromatography or radioimmunoassay) 4'6's'9,1113,16-18,24. In many cases only one dose of morphine was examined; in others a chronic injection or infusion schedule was used. Consequently, there is a wide variation of reported morphine concentrations for both blood and brain, as a function of dose or post-injection interval. Therefore, it is not unexpected that our values generally fall in the middle of this broad range. Our data are consistent with previous reports in showing that morphine concentration in all tissues is positively correlated with dose and negatively correlated with post-injection interval. The peak blood value in this study occurred at 30 min, which was the earliest time point measured. Other studies, particularly those using the intravenous route 6,17, obtained peak levels within 5-15 min. Because we did not take samples at earlier time points it is possible that peak blood levels might have occurred in less than 30 min. However, our data do show that, in intact rats, blood levels decline between 30 and 60 min, whereas morphine concentrations in the brain and spinal cord increase during that time, and then subsequently decline from 60 to 240 min. Unfortunately, we do not know whether spinal rats also show this difference in time course between peak morphine concentrations in the blood and CNS, because the earliest time point assayed in spinal rats was 60 min. These results clearly show, first, that at the dose and time points examined in this study blood morphine
concentration is not altered by acute spinal transection. These data suggest that spinalization does not disrupt the absorption of morphine into the circulation after subcutaneous injection. Second, the present results show that after subcutaneous injection morphine is not equally distributed between the brain and the spinal cord. At 60 min after an acute injection, approximately one-third of the total amount in the CNS is in the brain, and two-thirds are in the spinal cord. This differential distribution was consistent across all doses. Furthermore, the present data show that this relationship was not disrupted by spinal transection. Rats that were spinally transected 24 h previously had the same proportion of morphine in the brain and spinal cord as intact rats. However, although the distribution of morphine within the CNS was the same in intact vs spinal rats, the present data also show that the absolute amount of morphine at both sites in the CNS is significantly reduced by spinal transection. There was a significant decrease in morphine levels within the CNS across the doses and the time points examined in this study. These data are consistent with the experimental hypothesis that the decrease in behavioral opiate antinociception in acute spinal rats, after subcutaneous administration, is due to a decrease in spinal morphine concentration. The fact that morphine levels in both the brain and the spinal cord were reduced by spinalization whereas blood levels were not, suggests that the mechanism responsible involves a disturbance in (1) the integrity of the bloodbrain barrier; (2) blood flow within the CNS; or (3) both. The effects of spinal cord transection on the blood-spinal cord barrier have been examined and described at both the light and electron microscopic levels 15. These studies have found that there is disruption of the vasculature along the cord within 24 h after spinalization, and that it is more pronounced at regions that are 1.0-2.0 cm distal to the transection than at proximal sites. This demonstration of vascular damage suggests that spinalization might promote the entry of morphine into the CNS, or at least into the spinal cord. Such a prediction is not supported by the present data, since CNS morphine levels were reduced, rather than increased, after the transection. This outcome implies that the decrease in morphine levels is not simply a consequence of the trauma associated with spinalization, although the relationship between vascular damage and transport of morphine into the spinal cord remains to be empirically determined (e.g. see ref. 14). However, the results of a recent study by Sinclair et al. 19 also suggest that the decrease in CNS morphine levels may not simply be due to the physical trauma of spinalization. In that experiment, the TF reflex was elicited in intact rats after either systemic or intrathecal
258 m o r p h i n e injections. In each of these two conditions, the latency was m e a s u r e d before and after the spinal cord was reversibly cooled to 2 °C. These authors r e p o r t e d that the reversible cold block of the spinal cord decreased the antinociceptive effect of systemic morphine on the TF, but increased the antinociceptive effect of intrathecal morphine. This is the same response pattern seen in spinally transected rats. Presumably, a cold-block of the spinal cord does not d a m a g e spinal vasculature. On the o t h e r hand, this t r e a t m e n t might reduce blood flow, not only at the level of the spinal cord but throughout the CNS. A l t h o u g h it has been r e p o r t e d that spinal cord b l o o d flow is not i m p a i r e d after spinal transection, these measures were t a k e n only within the first 15 min after spinalization 23.
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Regardless of the mechanism involved, the fact that both spinalization and spinal cold block p r o d u c e d similar responses to both systemic and spinal morphine, is consistent with the present m o d e l , which proposes that in each of these situations, the effect of intrathecal morphine is enhanced because descending inhibitory control is r e m o v e d , and the effect of systemic morphine is reduced, because the m o v e m e n t of m o r p h i n e from the blood into the CNS is impaired. A d d i t i o n a l research will be required to u n d e r s t a n d the mechanism responsible for this impairment. Acknowledgements. This work was supported by USPHS Grant DA 02845. We thank J. Magoun, S. McCann and C. Mclnnis for technical assistance in performing the spinal transections and collecting the tissue samples.
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