Absence of appreciable tolerance and morphine cross-tolerance in rats with adrenal medullary transplants in the spinal cord

Neuropharmacology

0028-3908(94)EOOO5-C

Vol. 33, No. 5, pp. 681-692, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0028-3908/94-$7MJ

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Absence of Appreciable Tolerance and Morphine Cross-tolerance in Rats with Adrenal Medullary Transplants in the Spinal Cord H. WANG and J. SAGEN* Department of Anatomy and Cell Biology, University of Illinois at Chicago, 808 S. Wood Street, Chicago, IL 60612, U.S.A. (Accepted 20 December 1993)

Summary-Adrenal medullary transplants in the spinal subarachnoid space may be a means of achieving sustained local delivery of pain-reducing neuroactive substances on a continually renewable basis. However, a potential limitation of this approach is tolerance development to agents released from the transplanted cells. In particular, since adrenal medullary chromaffin cells release opioid peptides, reduced antinociceptive efficacy of opioids is possible. To determine this, alterations in the dose-effectiveness of morphine were assessed in animals with adrenal medullary transplants. Results indicated that, not only was there no apparent tolerance, but that adrenal medullary transplants could potentiate the analgesic efficacy of morphine. An additional goal of these studies was to determine whether chronic or intermittent nicotine could produce increased antinociception, since stimulation of cell surface nicotinic receptors increases release of neuroactive substances from chromaffin cells. This was assessed using subcutaneously implanted nicotine pellets or repeated systemic administration of nicotine. Findings indicated that exposure to nicotine results in an acute tolerance, or tachyphylaxis, to nicotine which is rapidly reversed following cessation of nitonic stimulation. Together, these results suggest that adrenal medullary transplants may provide a constant source of opioid peptides, augmentable by intermittent nicotinic stimulation, without the development of appreciable tolerance to these pain-reducing neuroactive substances. Keywordschromaffin nicotine.

cells, neural grafts, pain, analgesia, intrathecal, opioid peptides, spinal cord, tolerance,

nephrine and epinephrine), agents which independently reduce pain when injected directly into the spinal subarachnoid space. Furthermore, these agents appear to act synergistically such that the co-administration of subeffective doses of opioid and u-adrenergic agonists produces potent analgesia (Drasner and Fields, 1988; Sherman et al., 1988; Wilcox et al., 1987; Yaksh and Reddy, 1981). Previous work in our laboratory has shown that the adrenal medullary transplants in the rat spinal subarachnoid space can elicit significant antinociception to acute noxious stimuli, particularly following stimulation of cell surface nicotinic receptors, for at least 1 year following transplantation (Sagen and Pappas, 1987). This is most likely mediated, at least in part, by the release of both opioid peptides and catecholamines from the transplanted cells, since it can be attenuated by both opioid and a -adrenergic antagonists. Furthermore, such adrenal medullary transplants can produce long-term elevations in spinal CSF Met-enkephalin and catecholamine levels (Sagen and Kemmler, 1989; Sagen et al., 1991).

The transplantation of neurotransmitteror neurohormone-secreting tissues into the central nervous system (CNS) is a potentially powerful approach to altering CNS neurochemistry, and providing a continually renewable source of neuroactive substances for the alleviation of neural deficits. Work in our laboratory has suggested that transplants of adrenal medullary chromaffin cells into the spinal cord subarachnoid space may provide a constant source of pain-reducing agents for long-term pain relief as assessed by both acute noxious stimuli as well as chronic pain models, including neuropathic pain and inflammatory arthritis (Hama and Sagen, 1993; Sagen et al., 1986a, b, 1990). In particular, chromaffin cells synthesize and secrete opioid peptides (including Met- and Leu-enkephalin, as well as intermediate and larger-sized opioid peptides such as Metenkephalin-Arg-Phe, Met-enkephalin-Arg-Gly-Leu, and BAM-18; Boarder et al., 1982; Liston et al., 1984; Stern et al., 1980) and catecholamines (particularly norepi-

*To whom correspondence

should be addressed. 681

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Together, these results suggest that adrenal medullary transplants may provide a local and continually available source of opioid peptides and catecholamines for long-term pain relief, reducing or eliminating the need for repeated narcotic administration. However, since the transplanted cells constantly expose host spinal cord to pain-reducing neuroactive substances by releasing these agents on a continual basis, the potential for tolerance to the beneficial pain-reducing effects exists. In particular, tolerance to opioids, resulting in diminished analgesic potency, is a well-known and often frustrating result of repeated or continual administration. For example, using standard experimental analgesic assays such as the hot plate and tail flick tests in rodents, tolerance develops rapidly to repeated spinal or systemic morphine injections (Yaksh, 1984; Yaksh et al., 1977). Interestingly, while tolerance to spinal administration of morphine occurs rapidly in animal models, it is not always consistently observed, or develops at a slower rate in humans (Coombs et al., 1981; Zenz et al., 1985). The repeated administration of opioid peptides also results in rapid attenuation of their analgesic effects (Russell et al., 1987; Tseng, 1982; Yaksh et al., 1984). In addition, while endogenous opioid pentapeptides most likely act at the delta opioid receptor subtype, in contrast to morphine which appears more selective for the mu receptor, it has been suggested that there is some crosstolerance between mu- and delta-selective agonists at the spinal level (Russell et al., 1987; Tseng, 1982; Tseng, 1983; Yaksh, 1984). This issue may be particularly relevant to opioid mechanisms in adrenal medullary transplants, since chromaffin cells are thought to process several small, intermediate, and large-sized opioid peptides (Boarder et al., 1982; Liston et al., 1984; Stern et al., 1980), which may be active at both delta- and muopioid receptors (Hiillt, 1986). Finally, while numerous studies have suggested that spinally co-administered opioid and a -adrenergic agonists synergistically reduce nociception, there is also some indication of crosstolerance to a-adrenergic agents in animals tolerant to intrathecal morphine (Loomis et al., 1987a; Stevens et al., 1988). Together, the findings from these studies suggest that there is considerable potential for the development of tolerance to agents released by adrenal medullary transplants, which continually expose the host spinal cord to both opioid peptides and catecholamines (Sagen and Kemmler, 1989; Sagen et al., 1991). In addition, the possibility of cross-tolerance with exogenously administered opioids is not a trivial concern, as many chronic pain patients receive opioid therapy. Thus the goal of this study was to determine whether adrenal medullary transplants in the spinal subarachnoid space result in reduced analgesic efficacy or cross-tolerance to systemically administered morphine. In addition to basal release of opioid peptides and catecholamines from adrenal medullary transplants, it is possible to increase the release of these agents from

chromaffin cells by stimulating cell surface nicotinic receptors. Previous work in our laboratory has revealed potent antinociception to acute intense noxious stimuli in animals with adrenal medullary transplants following systemic injections of low doses of nicotine (Sagen et al., 1986a, b. 1991). In contrast, levels of neuroactive substances basally released from the transplanted cells are apparently sufficient for the reduction of chronic arthritic and neuropathic pain symptoms without nicotinic stimulation (Hama and Sagen, 1993; Sagen et al., 1990). These observations suggest that it is possible to increase the antinociceptive potency of adrenal medullary transplants by nicotinic stimulation, which may be useful for the management of acute intermittent episodes of more intense pain in chronic pain patients. However, it is well known that continual nicotinic stimulation of chromaffin cells results in rapid desensitization of nicotinic receptors, leading to the decreased secretion of neuroactive agents from the cells (Boksa and Livett, 1984; Malhotra et al., 1988; Schiavone and Kirpekar, 1982). Thus, another goal of this study was to determine the limitations in the use of nicotinic stimulation to enhance antinociceptive potency of adrenal medullary transplants. Daily administration of nicotine was used to assess whether intermittent stimulation would result in tolerance to the antinociceptive effects of nicotine in animals with adrenal medullary transplants. Subcutaneously implanted nicotine pellets were used to determine whether constant nicotine exposure would alter antinociception. A preliminary report of these studies has been presented previously (Wang and Sagen, 1989). METHODS

Surgical transplantation procedures

Adult male Sprague-Dawley rats (Sasco Inc., WI) weighing 300-350 g were used as both donors and recipients. Adrenal medullary tissue was prepared and transplanted into the spinal subarachnoid space at the level of the lumbar enlargement as described previously (Sagen et al., 1986a, 1990). Adrenal medullary tissue from two adrenal medullae was transplanted, as this has been shown to reliably produce antinociception in previous studies (Sagen et al., 1986a, 1990; Sagen and Pappas, 1987). Control animals received equal volumes of striated muscle tissue using identical implantation procedures. Animals were allowed a minimum of 4 weeks for recovery from surgical procedures, and stabilization of graft integration before behavioral testing. Analgesiometric testing procedures

To assess changes in responses to noxious stimuli, three analgesiometric tests were used sequentially: the tail flick test, paw pinch test (Randall-Selitto), and hot plate test, as described previously (Sagen et al., 1986a, b). In order to prevent tissue damage in the absence of a response, the beam is automatically termi-

Tolerance and adrenal medullary implants nated at 14 set, and the animal assigned a latency of 14. Since there is some controversy regarding the role of tail skin temperature in the tail flick latencies which indicate a possible inverse correlation between the two variables (Berge et al., 1988), tail skin temperatures were measured in preliminary studies using an infrared thermometer (Kent Scientific). The paw pinch response was elicited by a commercially available apparatus (Ugo-Basile) that applies pressure at a constant increasing rate of 64 g/set, and automatically terminates at a scale reading of 25 (1000 g). The hot plate response was determined by the latency to licking or biting the hind paws, and terminated at 40 set in the absence of a response. This test was included as a complementary test to the reflexive models, as it involves a more complex integrated response reflecting processing at higher levels (Hammond, 1989; Ramabadran and Bansinath, 1986). However, due to some apparent learning after repeated exposure to the hot plate, which results in a jumping escape response immediately after placement in the hot plate apparatus, this test could not be used in studies that required repeated testing within a short time frame (e.g. >4 times/month). Experimental

design

Dose-responsiveness to morphine. In order to estimate the potential development of tolerance to opioids released from the transplants, dose-responsiveness to morphine was assessed in rats with adrenal medullary or control muscle implants prior to and following transplantation. A systemic route of morphine delivery was chosen, as it is the most common route of morphine administration in the clinical setting. Prior to transplantation, baseline nociceptive thresholds, and response to morphine sulfate (1.25, 2.5 and 5.0 mg/kg, s.c.) 20 min after injection were assessed using the tail flick, paw pinch and hot plate tests (n = 40). Testing took place at 5 day minimum intervals on a rotating dose schedule, such that approximately one third of the animals received each of the doses on each test date, and all of the animals eventually received all three doses. The animals were then randomly divided into two groups, and implanted with either adrenal medullary tissue (n = 20) or control tissue (n = 20). Dose-responsiveness to morphine was again assessed on a similar schedule 4-6 weeks following transplantation. Chronic exposure to nicotine. To determine the effect of continuous nicotine exposure on antinociceptive responses to acute nicotinic stimulation, subcutaneous implanted constant delivery nicotine pellets were used (see below for details). Dose-responsiveness to nicotine was assessed in adrenal medullary implanted animals before pellet implantation, during steady state exposure with nicotine pellets, and following pellet depletion using the tail flick and paw pinch tests. The hot plate test could not be included in this study, since the required repeated

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testing resulted in an apparent learning phenomenon, as animals jumped immediately after placement in the hot plate chamber. Prior to pellet implantation, nociceptive responses in animals with adrenal medullary transplants (n = 48, to allow for 8 animals/pellet dose below) were determined at three doses of acute nicotine administration (free base, 0.05, 0.1, 0.2 mg/kg, s.c.) at 2 min following injection (since previous studies have shown that this is the time of peak antinociceptive effect; Sagen et al., 1986a, b). Testing occurred at 5 day minimum intervals on a rotating dose schedule, such that approximately one third of the animals received each of the doses on each test date, and all of the animals eventually received all three doses. For comparison, nociceptive responses in animals with control transplants were also determined at these three doses, although previous studies have revealed that these doses do not significantly alter responses in control animals (Sagen et al., 1986b, 1994). Nicotine pellets (Innovative Research of America) were used to deliver a constant level of nicotine to produce a continuous exposure. Pellets were implanted subcutaneously via a small incision in the neck. These pellets contain various amounts of nicotine (0.625, 1.25, 2.5,5.0 and 10.0 mg) which is delivered over a three week period. Steady state nicotine levels attained by these pellets are 0.01,0.02, 0.05, 0.1 or 0.2 mg/kg, respectively (T,,2 = 2 hr). As a control, placebo pellets of equal size with no nicotine were used. Animals with adrenal medullary transplants were randomly divided into 6 groups and were implanted with nicotine (0.625, 1.25, 2.5, 5.0 or lO.Omg) or placebo pellets (n = 8/pellet size). Beginning one week following pellet implantation, nociceptive responses to acute nicotine injections (0.05, 0.1 and 0.2 mg/kg, s.c.) were again assessed using a schedule similar to that described above. Finally, to determine whether constant exposure effects were reversible, nociceptive responses to acute nicotine injections were again assessed 3 weeks following depletion of nicotine pellets. In order to determine whether antinociceptive effects of morphine are altered in adrenal medullary transplanted animals with constant exposure to nicotine, another group of animals was tested with morphine (1.25, 2.5 and 5.0mg/kg, s.c.) before and one week following implantation of similar sized nicotine pellets (8 animals/pellet dose). Repeated exposure to nicotine. For assessment of repeated exposure to nicotine, some animals with adrenal medullary transplants (n = 12) were injected (nicotine, 0.1 mg/kg, s.c., daily) and tested daily for 12 days. In addition, in order to determine the recovery period necessary for a full antinociceptive response to nicotine, another group was given a second acute injection of nicotine at several time intervals following an initial nicotine injection. Baseline nociceptive responses and responses 2 min following nicotine injection (0.1 mg/kg, s.c.) were assessed in a group of animals with

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adrenal medullary transplants (n = 40). Animals were randomly divided into 5 groups which were challenged with a second injection of nicotine at different intervals (0.5, 1.0, 2.0, 4.0 and 6.0 hr). For the second trial, baseline nociceptive responses, and response to nicotine injection (0.1 mg/kg, s.c.) were again determined. Statistical analysis

Statistical comparisons between treatment groups were done using two-way analysis of variance and the Newman-Keuls test for multiple post-hoc comparisons. In addition, in order to assess changes in morphine antinociceptive potency in transplanted animals, potency ratios, A50s and 95% confidence limits were calculated (Tallarida and Murray, 1987). Morphological analysis

temperature studies.

interference

in tail flick latencies in our

Dose -responsiveness to morphine

Figure 1 shows the response to morphine (1.25, 2.5 and 5.0 mg/kg, s.c.) before and 4-6 weeks after transplantation of either adrenal medullary or control muscle tissue. Before transplantation, systemic injections of morphine produced dose-related increases in tail flick latency, paw pinch threshold and hot plate latency. A reduction in the antinociceptive efficacy of morphine would be expected to result in a rightward shift of this morphine dose-response curve. Results indicated that the transplantation of control muscle tissue did not significantly alter response to morphine compared to pre-transplantation values as assessed by any of the three analgesiometric tests (P > 0.05 for all three tests). In

Following termination of behavioral studies, the viability of chromaffin cells in adrenal medullary transplants was assessed using immunocytochemistry. Animals were deeply anesthetized (50 mg/kg pentobarbital), and perfused via the ascending aorta with saline, followed by fixative (4% paraformaldehyde in 0.1 M phosphate buffer). Spinal cord regions containing the transplants were removed, cryoprotected overnight in 20% sucrose, and sectioned on a cryostat (HackerBright). Fifteen micron sections were mounted on gelatin-coated glass slides, incubated in blocking agent (2.0% normal goat serum in phosphate-buffered saline), and exposed to primary antisera (mouse anti-tyrosine hydroxylase, Incstar, diluted 1:500) overnight at 4°C. Some sections were incubated in pre-immune serum only as a control. Sections were then washed and exposed to secondary antisera (rhodamine-conjugated goat-antimouse, Organon, diluted 1:lOO) for 1 hr at room temperature. Sections were again washed, coverslipped in Fluoromount, and viewed in a Zeiss Axiophot epifluorescence microscope.

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RESULTS Tail skin temperatures

Tail skin temperatures were measured in preliminary studies due to the suggestion that there may be an inverse relationship between tail flick latencies and skin temperatures which could interfere with the interpretation of the results (Berge et al., 1988). Findings revealed no apparent correlation between skin temperature and tail flick responses. Neither graft implantation nor chronic nicotine pellet exposure produced significant alterations in tail skin temperatures. In addition, while acute nicotine injections in adrenal medullary transplanted animals produced increased tail flick latencies in adrenal medullary but not control transplant groups, tail skin temperatures were slightly elevated in all animals, with no significant difference between adrenal medullary and control transplant groups. This effect is opposite to the predicted inverse correlation, arguing against skin

8J

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Fig. 1. Dose-response curves to morphine in animals before and after adrenal medullary or control transplants. The ordinate is the threshold for response to noxious stimuli (mean f SEM) as assessed by the tail flick test (top), paw pinch test (middle), and hot plate test (bottom) 20 min following morphine injection. The abscissa represents doses of acute morphine injections (1.25, 2.5 and 5.0 mg/kg, s.c.) in animals before (0; n = 40) and 4-6 weeks after adrenal medullary (A; n = 20) or control (A; n = 20) transplants in the spinal subarachnoid space.

Tolerance and adrenal medullary implants contrast, dose-response curves to acute systemic morphine were shifted significantly to the left in animals with adrenal medullary tissue implanted in the spinal subarachnoid space as assessed by both tail flick (P < 0.01) and paw pinch tests (P -C 0.05; Fig. 1, top and middle

panels). Rather than the predicted rightward shift indicative of reduced morphine efficacy, these results indicate a potentiation in morphine dose-effectiveness. A rightward shift in the dose-response to morphine assessed by hot plate test also did not occur, as there were no significant alterations in hot plate responsiveness in adrenal medullary transplanted animals (P > 0.05; Fig. 1, bottom panel). These results indicate that there is no apparent decrement in the antinociceptive potency of systemically administered morphine in animals with adrenal medullary tissue chronically implanted in the spinal cord subarachnoid space. The calculated A50 and 95% confidence limit values as assessed by the tail flick test for morphine in adrenal 0

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medullary implanted animals were 1.89 mg/kg [ 1.6 1, 2.201 compared to 3.05 mg/kg [2.57, 3.631 before transplantation and 2.83 mg/kg [2.42, 3.311 after control transplants. The values in both pre-transplanted and control-transplanted animals were similar to those previously reported (Fleetwood and Holtzman, 1989; Hammond, 1989), but slightly higher than those reported by others (Zhang and Pasternak, 1981). Calculated potency ratios were 1.96 and 1.03 for adrenal medullary and control transplants, respectively, compared to pre-transplantation values. As assessed by the paw pinch test, calculated A50 and 95% confidence limit values were 0.47 mg/kg [O.12, 0.871 in adrenal medullary implanted animals compared to 1.31 mg/kg [0.94, 2.891 before transplantation and 1.29 mg/kg [0.89, 2.441 after control transplants. Potency ratios were 2.61 and 1.16 for adrenal medullary and control transplants, respectively, compared to pre-transplant values. Since the dose-response to morphine as assessed by the hot plate was not significantly altered by adrenal medullary transplants, A50s and potency ratios were not calculated. Chronic exposure to nicotine

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Fig. 2. Dose-responsiveness to nicotine in animals with adrenal medullary transplants following implantation of nicotine pellets. The ordinate is the threshold for response to noxious stimuli as assessed by the tail flick test (top panel) or paw pinch test (bottom panel). Points represent nociceptive responses (mean f SEM) 2 min following acute injections of nicotine (0.05, 0.1 and 0.2 mg/kg, s.c.) in animals with adrenal medullary transplants following subcutaneous implantation of several doses of nicotine pellets or placebo pellets (n = 7-8 animals/pellet dose). For comparison, the dose-response to nicotine in animals with control striated muscle transplants is included. The abscissa represents acute nicotine injection dose.

Chronic exposure to nicotine was accomplished by implantation of nicotine releasing pellets. Animals were tested before pellet implantation, during the three week pellet period, and three weeks after pellet depletion. Both dose-responsiveness to acute injections of nicotine (0.05, 0.1, 0.2 mg/kg, s.c.) and morphine (1.25, 2.5, 5.0 mg/kg, s.c.) were assessed in separate groups using the tail flick and paw pinch tests. The effects of pellet implantation on the antinociceptive efficacy of nicotine injections are shown in Fig. 2. Results indicated that the implantation of placebo pellets did not significantly alter dose-responsiveness to acute systemic nicotine in rats with adrenal medullary implants compared to pre-pellet values (P > 0.05). However, the larger size nicotinic pellets, such as 2.5, 5.0 and 10.0 mg (which deliver steady state nicotine levels of 0.05, 0.1 and 0.2 mg/kg, respectively), flattened the dose-response curves to acute systemic nicotine compared to placebo pellets (Fig. 2, P c 0.01 for both tests). This reduced antinociceptive efficacy was so marked in adrenal medullary implanted animals with 2.5, 5.0 and lO.Omg pellets that nociceptive thresholds following acute nicotine injections were not significantly different than those in control implanted animals (P > 0.05). In contrast to the larger pellets, the lowest dose nicotinic pellets (0.625 mg size; 0.01 mg/kg steady state) did not significantly alter dose-response curves to acute systemic nicotine in adrenal medullary transplants compared to placebo pellets (P > 0.05 for both tests). An intermediate level produced by the 1.25 mg (0.02 mg/kg steady state) pellets produced variable effects, depending on the test (P > 0.05, tail flick; P < 0.05, paw pinch; compared to placebo pellets). The reduced antinociceptive efficacy of acute nicotine injections in adrenal medullary implanted animals was observed during the l-2 weeks following implantation of

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the larger dose nicotinic pellets. To assess recovery of response to nicotine in animals with transplants, antinociceptive responsiveness to acute nicotine injections were again assessed 3 weeks following depletion of nicotine from the subcutaneously implanted pellets. Antinociceptive responses to acute injections (nicotine, 0.1 mg/kg, s.c.) before adrenal medullary transplan-

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tation, after adrenal medullary transplantation, after nicotinic pellet implantation, and after depletion of nicotine from the pellets are shown in Fig. 3. Similar to previously reported results (Sagen et al., 1986a, 1990), this dose of nicotine does not significantly alter nociceptive responses in unimplanted animals (clear bars in Fig. 3). However, following transplantation of adrenal medullary tissue, nicotine injection resulted in marked antinociception as assessed by both tail flick and paw pinch tests (diagonal bars in Fig. 3). Responses to acute nicotine injections during nicotine pellet release are shown by the filled bars in Fig. 3. As described above,

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Fig. 3. Analgesiometric responses to nicotine in rats with spinal subarachnoid space adrenal medullary transplants before, during, and after subcutaneous nicotine pellet implantation. The ordinate is the threshold for response to painful stimuli assessed by the tail flick test (top) and paw pinch test (bottom) 2 min following systemic nicotine injections (0.1 mg/kg, se.). Bars represent the mean f SEM nociceptive responses to nicotine before adrenal medullary implantation (open bars), 4-6 weeks following adrenal medullary implantation prior to pellet implantation (diagonal bars), during nicotine pellet implantation (filled bars), and following depletion of nicotine pellets (cross-hatched bars). The abscissa shows the size of the implanted nicotine pellets (n = 7-8 animals per pellet dose).

5.0

2.5

(mg/kg)

Fig. 4. Dose-response to morphine injections in rats with spinal subarachnoid space adrenal medullary transplants after subcutaneous nicotine pellet implantation. The ordinate is the threshold for response to noxious stimuli (mean f SEM) as assessed by the tail flick test (top) and paw pinch test (bottom) 30 min following systemic morphine injections (1.25, 2.5 and 5.0 mg/kg, s.c.). Points represent mean +SEM nociceptive responses during implantation of various doses of nicotine pellets or placebo pellets. The abscissa is the dose of systemic morphine injections (n = 6-8 animals per pellet dose).

the implantation of placebo pellets did not significantly alter response to nicotine in these animals (P > 0.05). In contrast, the implantation of nicotine-pellets, particularly the larger sizes (2.5, 5.0 and 10.0 mg) reduced antinociceptive responses to acute nicotine in both analgesiometric tests. In animals with 2.5, 5.0 and lO.Omg pellets, responses to nicotine were reduced to pre-adrenal transplantation levels (P > 0.05), and significantly reduced from pre-pellet values (P < 0.01). Six weeks after the implantation of nicotine pellets (when nicotine had been depleted; cross-hatched bars in Fig. 3), antinociceptive responses to acute nicotine as assessed by tail flick and paw pinch were almost recovered to the levels of those before the pellet implantation in all groups (P > 0.05 compared to pre-pellet value). At this point, acute nicotine injections again produced significant analgesia in animals with adrenal medullary transplants as assessed by tail flick and paw pinch tests (P -C0.01). These results indicate that the constant delivery of high levels of nicotine can reduce antinociceptive

687

Tolerance and adrenal medullary implants potency of acute nicotinic injections in animals with adrenal medullary transplants in the spinal subarachnoid space. In contrast to the reduced efficacy of nicotine, dose-response curves to acute systemic morphine were not altered by the implantation of nicotine pellets in adrenal medullary implanted animals (Fig. 4). Even the largest nicotine pellets failed to significantly reduce the antinociceptive efficacy of morphine in these animals (P > 0.05 compared to placebo pellets). Interestingly, potentiation of morphine antinociceptive efficacy comparable to that observed earlier (see Fig. 1) appeared to be retained in these adrenal medullary transplanted animals, even in the presence of nicotine pellets. Repeated exposure to nicotine

To determine whether acute nicotine could be given on a daily basis, some animals with adrenal medullary implants were tested daily for two weeks with acute systemic nicotine injections (0.1 mg/kg, s.c.). No significant decrements in the antinociceptive response (Fig. 5, P > 0.05) were found following daily injections of nicotine in adrenal medullary implanted animals. These results indicate that antinociceptive responses to acute nicotine injections in adrenal medullary implanted animals can recover within 24 hr, and that the daily administration of nicotine does not result in the apparent induction tolerance to nicotine. However, since the previous findings indicated that chronic exposure to nicotine can reduce the antinociceptive efficacy of nicotine in these animals, it is likely that there is some form of acute tolerance, or tachyphylaxis, which is recoverable following cessation of exposure to nicotine. To assess the time interval necessary for recovery of the nicotinic response in animals with adrenal medullary transplants, a second acute nicotine injection was given at several intervals following an initial nicotine injection. Results are shown in Fig. 6. -Both baseline antinociceptive responses and responses following nicotine (0.1 mg/kg, s.c.) are shown. As previously demonstrated, an initial 12 ‘;; 3

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rig. 0. iacnypnyiaxis to acute nicotine injections in animals with adrenal medullary transplants. The ordinate indicates nociceptive threshold (mean f SEM) as assessed by the tail flick (top), paw pinch (middle), and hot plate (bottom) tests. Baseline nocieeptive responses and responses 2 min following an initial systemic injection of nicotine (0.1 mg/kg, s.c.) are shown by the diagonal and open bars, respectively. Baseline responses and responses 2 min following a second injection of nicotine (0.1 mg/kg, s.c.) are shown by the cross-hatched and filled bars, respectively. The abscissa represents the time interval after first acute nicotine injections (n = 7-8 animals/group).

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Fig. 5. Tail flick response induced by daily nicotine injections in animals with adrenal medullary transplants. The ordinate is the threshold nocieeptive response (mean + SEM) before (filled bars) and 2min following systemic nicotine injection (0.1 mg/kg, s.c.). The ordinate indicates days of repeated nicotine injection (n = 12 animals).

injection of nicotine produced significant antinociception on all three analgesiometric tests in adrenal medullary transplanted animals (P < 0.01, clear bars in Fig. 6). At 0.5, 1.O, 2.0, 4.0 and 6.0 hr following initial analgesiometric testing, baseline levels of pain thresholds on all three tests were recovered to pre-injection values (P > 0.05, cross-hatched bars in Fig. 6). However, the antinociceptive responses to a second injection of nicotine were altered depending on the time interval from the first injection. When the second nicotine injection

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was delayed for 4 hr or greater after the first injection, antinociceptive effects were significant (P < 0.01 compared to baseline) and were the same as those induced by the first nicotine injections (P > 0.05 compared to the first nicotine injection, solid bars in Fig. 6). However, with inter-injection intervals less than 4 hr, reduced responses were obtained. Following an interval of 2 hr, antinociceptive significant effects were obtained (P < 0.05), but the levels of antinociception were lower than the levels induced by first nicotinic stimulation (P < 0.05 for tail flick and paw pinch tests). With the second nicotine injections at intervals of 0.5 or 1.0 hr following the first trials, no significant antinociceptive effects were elicited (P > 0.05 compared to baseline), except for the tail flick (P < 0.05 at 1 hr). Morphological Jindings

Following termination of behavioral testing procedures, all adrenal medullary transplants were analyzed immunocytochemically to confirm chromafhn cell survival in adrenal medullary transplants. Figure 7 shows a typical adrenal medullary transplant in the rat spinal subarachnoid space as it appears 10 weeks following transplantation. The chromaffin cells are densely clustered in the adrenal medullary graft, located dorsal to the host spinal cord in the subarachnoid space. The cells appear to be healthy, and generally retain morphological characteristics found in the in situ adrenal medulla such as a cuboidal shape with little evidence of sprouting or differentiation. Note that there is no apparent gross disruption or compression of host spinal tissue, and that

the graft appears to reside in the spinal CSF loosely attached to the meningeal surfaces. DISCUSSION The present findings and results from previous studies in our laboratory demonstrate that adrenal medullary tissue transplanted into the subarachnoid space of the spinal cord can reduce nociception. Morphologic analyses suggest that these transplants act via release of neuroactive substances in the spinal CSF rather than via direct synaptic integration with host neural circuitry, since there is little evidence of host-graft integration. This is further supported by neurochemical studies which reveal increased levels of catecholamines and opioid peptides in the spinal CSF following adrenal medullary implantation (Sagen and Kemmler, 1989; Sagen et al., 1991). Together, these data suggest that adrenal medullary transplants reduce nociception by providing a continually renewable source of painreducing neuroactive substances locally in the spinal subarachnoid space. A potential limitation in the use of neural transplants for pain alleviation is the possible development of tolerance, or reduced analgesic efficacy, with constant exposure to pain-reducing neuroactive substances. Previous studies in our laboratory have suggested that tolerance development to basally released levels of neureactive substances from adrenal medullary transplants is not a significant problem, since these transplants continue to alleviate chronic pain behaviors in both

Fig. 7. Appearance of an adrenal medullary tissue 10 weeks following transplantation in the spinal subarachnoid space. Chromaffin cells in the transplant are identified by a tyrosine hydroxylase antibody labeled with a rhodamine-linked secondary antibody.

Tolerance and adrenal medullary implants arthritic and neuropathic pain models during the entire course of the syndromes (Hama and Sagen, 1993; Sagen et al., 1990). In addition, results of the present study suggest that daily exposure to increased levels of substances released by chromaffin cells also does not produce appreciable tolerance, since daily stimulation of chromaffin cell release by nicotine does not result in a significant decrement in antinociceptive potency. This is in contrast to findings with repeated systemic or intrathecal opioid administration, which results in the rapid development of diminished analgesic efficacy (Yaksh, 1984; Yaksh et al., 1977). Thus, neither chronic basal levels nor intermittently increased levels of substances released from transplanted chromaffin cells result in apparent tolerance to antinociceptive effects. A possible explanation for this apparent lack of tolerance is the short half-life of natural analgesic substances released from the chromaffin cells due to rapid metabolism or clearance, which limits exposure to host spinal receptors. In support for this, previous findings in our laboratory have suggested that the concentrations of opioid peptides and catecholamines released from transplanted adrenal chromaffin cells, while significantly increased compared to control animals, are probably too low to produce potent antinociception in isolation (Sagen and Kemmler, 1989; Sagen ef al., 1990). Rather, it is likely that the combined release of agents acting at both opioid and a-adrenergic receptors reduce nociception by a synergistic mechanism. The synergistic antinociceptive activity with co-administration of subeffective doses of opioid and a-adrenergic agonists has been well documented (Drasner and Fields, 1988; Sherman et al., 1988; Wilcox et al., 1987). Furthermore, Yaksh and Reddy (198 1) demonstrated that such combinations administered intrathecally resulted in no apparent decrement in antinociceptive potency, in contrast to the rapid tolerance development with intrathecal morphine, and suggested that this was due to a minimum degree of activation of either receptor. Thus, the lack of apparent tolerance in adrenal medullary implanted animals may be due to the co-activation of opioid and GI-adrenergic receptors with subeffective levels of both classes of agents. In addition to the lack of apparent tolerance to both basal and stimulated release of neuroactive substances from transplanted chromaffin cells, there is also no decrement in the antinociceptive potency of systemically administered morphine. This is somewhat surprising, since numerous studies have demonstrated crosstolerance between systemically- and spinally-administered opioids (Delander and Takemori, 1983; Tyler and Advokat, 1986; Yaksh et al., 1977). However, this cross-tolerance may be asymmetric, since tolerance to systemic morphine is not consistently observed in animals rendered tolerant to chronic intrathecal morphine (Advokat et al., 1987; Loomis et al., 1987b). In the present study, there was no apparent tolerance to systemic morphine in animals with adrenal medullary im-

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plants in the spinal subarachnoid space, but whether or not the converse is true (i.e. reduced development of morphine tolerance in implanted animals) awaits further studies. Another possible explanation for the lack of cross-tolerance to morphine in adrenal medullary implanted animals is that different opioid receptor subtypes are involved. In particular, the pentapeptides Met- and Leu-enkephalin possess a high selectivity for the delta receptor, while morphine likely acts primarily at mu receptors (Garzon et al., 1983; Lord et al., 1977; Yaksh, 1987). Thus, continuous activation of spinal delta receptors by adrenal medullary transplants may not necessarily alter responses to mu agonists. However, it has been suggested that there is some cross-tolerance between mu- and delta-selective agonists in animals rendered tolerant to spinally administered delta agonists (Russell et al., 1987; Tseng, 1982, 1983; Yaksh, 1984). Further, the processing of proenkephalin in the adrenal medulla most likely results in several intermediate and large-sized opioid peptides, in addition to Met- and Leu-enkephalin, which may be active at the mu site (Broader et al., 1982; Hollt, 1986; Liston et al., 1984; Stern et al., 1980). For example, the heptapeptide Metenkephalin-Arg-Phe and the octapeptide Met-enkephalin-Arg-Gly-Leu have comparable affinities for the mu and delta receptors, and derivatives of the peptide E domain such as BAM- 18 are highly selective for the mu receptor, and have significant antinociceptive potency (Iadorola et al., 1986; Inturrisi et al., 1980; Stevens et al., 1988). Thus, both delta and mu receptors are likely to be activated by substances released by adrenal medullary transplants. This may account for the potency of adrenal medullary transplants, since mu-preferring peptides appear to be stronger antinociceptive agents than deltapreferring peptides (Goodman and Pasternak, 1984). Further, agents having high affinity for both mu and delta receptors are among the most active (Yaksh, 1987). Interestingly, there is evidence for a synergistic interaction between mu and delta opioid agonists in antinociception (Larson et al., 1980; Porreca et al., 1990) although other studies have found low mu-delta interactions (Sanchez-Blazquez and Ganzon, 1993). In addition to activation of mu and delta opioid receptors, it is possible that kappa opioid receptor activation contributes to the antinociception produced by adrenal medullary transplants. Although prodynorphin products are low in the rat adrenal medulla (Evans et al., 1988), some proenkephalin-derived products such as Met-enkephalin-Arg-Phe may also be active at the kappa receptor. In support for this, recent findings in our laboratory using specific opioid antagonists have indicated that all three receptor subtypes must be blocked in order to completely attenuate antinociception by adrenal medullary transplants (Wang and Sagen, 1993). A potential synergism between multiple opioid receptor activation could also account for the observed

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potentiation of morphine antinociception in animals with adrenal medullary transplants revealed in the present study. Not only was there no apparent cross-tolerance to systemic morphine, but dose-response curves were actually shifted left on the tail flick and paw pinch tests, indicative of a potentiation of morphine. Similarly, the administration of subeffective doses of Leu-enkephalin can reduce the analgesic ED50 of morphine, and shift the morphine dose-response curve leftward (Larson et al., 1980; Porreca et al., 1990). Furthermore, the corelease of catecholamines from transplanted chromaffin cells could produce an additive or synergistic antinociception with symmetrically administered morphine, by co-activation of opioid and a-adrenergic receptors, as described above. Finally, chromaffin cells produce and release several other neuroactive substances, e.g. neuropeptide Y, which have antinociceptive properties and could potentiate morphine antinociception (Hua et al., 1991). As adrenal cortical cells cannot be completely eliminated from the transplants, the role of agents released from these cells, such as glucocorticoids, also cannot be discounted. Thus, results of this study indicate that, not only is there no apparent tolerance to opioids released from adrenal medullary transplants, but that the transplants can increase the antinociceptive efficacy of systemically administered morphine. Another aim of this study was to determine whether transplanted chromaffin cells can be continually stimulated to release increased levels of neuroactive substances via prolonged activation of cell surface nicotinic receptors. Results indicated that the constant exposure to high levels of nicotine can reduce the antinociceptive efficacy of nicotinic stimulation in adrenal medullary implanted animals. Additionally, diminished responsiveness was also observed for at least 2 hr following acute nicotine administration, with recovery of full analgesic efficacy within approx 4 hr. These results could indicate that chronic nicotine stimulation results in tolerance to prolonged exposure to high levels of pain-reducing neuroactive substances released by the transplanted cells, particularly opioid peptides. However, this is unlikely since chronic nicotine exposure did not alter responsiveness to morphine in animals with adrenal medullary transplants. In fact, the morphine dose-response remained potentiated in these animals even during high levels of nicotine exposure, arguing against tolerance via prolonged increased release of opioid peptides from the transplants. The most likely explanation for the reduced antinociceptive potency of nicotine during chronic exposure is the desensitization of chromaffin cell surface nicotinic receptors, resulting in diminished release of neuroactive substances from the transplanted cells. The decline in catecholamine release from adrenal medullary chromaffin cells in the continued presence of cholinergic agonists such as nicotine had been well established (Boksa and Livett, 1984; Malhotra et al., 1988; Schiavone and Kirpekar, 1982). The desensitization of nicotinic receptors occurs rapidly during

nicotine exposure in vitro (within 5 min). In addition, recovery from nicotinic desensitization is also fairly rapid following removal of nicotine (approx 30 min). Similarly, findings of the present study demonstrated recovery of the full analgesic response within 4 hr (2 x the T,,*) following acute administration of nicotine. Thus, it is likely that cell surface receptors on transplanted chromaffin cells are rapidly desensitized by acute or chronic exposure to nicotine, and that recovery occurs following cessation of nicotinic exposure. These results suggest that chronic nicotine delivery (e.g. via a nicotine skin patch) would not be an optimal means for producing antinociception with adrenal medullary transplantation, but that intermittent stimulation of nicotinic receptors may be useful in reducing acute episodes of more intense pain, if nicotinic side effects could be minimized. The development of tolerance to nicotine administration in the present study may also be due to changes in host responses to nicotine, since high doses of nicotine have been shown to produce antinociception (Sahley and Berntson, 1979; Tripathi et al., 1982) and chronic exposure can produce tolerance to an acute challenge of this drug (Collins et al., 1988). Although the mechanisms that underlie tolerance to nicotine are not well understood, recent evidence suggests that chronic nicotine treatment results in an up-regulation of brain nicotinic receptors with a concomitant receptor desensitization or inactivation (Collins et al., 1990). Therefore, in the present study, tolerance to implanted high doses of nicotinic pellets may be due to changes in host nicotinic receptors. In summary, the results of this study indicate that there is little apparent tolerance to agents chronically released by chromaffin cells transplanted to the spinal cord subarachnoid space, although augmentation of release by nicotinic stimulation is accompanied by a short-lived tachyphylaxis. In addition, the antinociceptive efficacy of morphine appears to be enhanced by adrenal medullary transplants. Previous studies in our laboratory using chronic pain models reveal no decrement in paid-reducing effects of adrenal medullary transplants in the absence of nicotinic stimulation (Hama and Sagen, 1993; Sagen et al., 1990). Together, results from the present and previous studies suggest that basal release of opioid peptides and catecholamines from adrenal medullary transplants may be sufficient for alleviation of chronic pain, and that augmentation by nicotinic receptor stimulation or supplementation with exogenous opioids could be useful during acute episodes of more intense pain. Thus, adrenal medullary transplants in the spinal subarachnoid space may provide a continually renewable source of pain-reducing neuroactive substances for the alleviation of pain without the development of appreciable tolerance. The animal studies were done in accordance with the guidelines for the care and use of laboratory animals established by the National Institutes of Health and the

Tolerance and adrenal medullary implants Animal Care Committee at the University of Illinois at Chicago. All efforts were made to minimize animal suffering and the number of animals used. Acknowledgement-This NS25054.

work was supported by NIH Grant

REFERENCES Advokat C., Burton P. and Tyler C. B. (1987) Investigation of tolerance to chronic intrathecal morphine infusion in the rat. Physiol. Behav. 39: 161-168. Berge 0. G., Garcia-Cabrera I. and Hole K. (1988) Response latencies in tail-flick test depend on tail skin temperature. Neurosci. Lett. 86: 284-288.

Boarder M. R., Lockfeld, A. J. and Barchas J. D. (1982) Met-enkephalin [Arg6,Phe7] immunoreactivity in bovine caudate and bovine adrenal medulla. J. Neurochem. 39: 149-I 54.

Boksa P. and Livett B. G. (1984) Desensitization to nicotinic cholinergic agonists and K+, agents that stimulate catecholamine secretion, in isolated adrenal chromaffin cells. J. Neurochem. 42: 607-617.

Collins A. C., Bhat R. V., Pauly J. R. and Marks M. J. (1990) Modulation of nicotine receptors by chronic exposure to nicotinic agonists and antagonists. Ciba Found. Sympos. 152: 68-86.

Collins A. C., Romm E. and Wehner J. M. (1988) Nicotine tolerance: an analysis of the time course of its development and loss in the rat. Psychopharmacology %: 7-14. Coombs D. W., Saunders R. L., Gaylor M. S., Pageau M. D., Leith M. G. and Schaiberger C. (198 1) Continuous epidural analgesia via implanted morphine reservoir. Luncet 2: 425-426.

Delander G. E. and Takemori A. E. (1983) Spinal antagonism of tolerance and dependence induced by systemically administered morphine. Eur. J. Pharmac. 94: 3542. Drasner K. and Fields H. F. (1988) Synergy between the antinociceptive effects of intrathecal clonidine and systemic morphine in the rat. Pain 32: 309-312. Evans C. M., Hammond D. L. and Frederickson R. C. A. (1988) The opioid peptides. In: The Opiate Receptors (Edited by Pasternak G. W.), pp. 23-71. Humana Press, New Jersey. Fleetwood S. W. and Holtzman S. G. (1989) Stress-induced potentiation of morphine-induced analgesia in morphinetolerant rats. Neuropharmacology 28: 563-567. Garzon J., Jen M. F., Sanchez-Blazquez P., Hollt, V., Lee N. M. and Loh H. H. (1983) Endogenous opioid peptides: comparative evaluation of their receptor affinities in the mouse brain. Life Sci. 33 (suppl. 1): 291-294. Goodman R. R. and Pasternak G. W. (1984) Multiple opiate receptors. In: Analgesics: Neurochemical, Behavioral, and Clinical Perspectives (Kuhar M. and Pasternak G., Eds), pp. 69-96. Raven Press, New York. Hama A. T. and Sagen J. (1993) Reduced pain-related behavior by adrenal medullary transplants in rats with experimental painful peripheral neuropathy. Pain 52: 223-231. Hammond D. L. (1989) Inference of pain and its modulation from simple behaviors. In: Issues in Pain Measurement (Chapman C. R. and Loeser J. D., Eds), pp. 69-91. Raven Press, New York. Hollt V. (1986) Opioid peptide processing and receptor selectivity. A. Rev. Pharmac. Toxic. 26: 59-77. NP3,,5--E

691

Hua X.-Y., Boublik J. H., Spicer M. A., Rivier J. E., Brown M. R. and Yaksh T. L. (1991) The antinociceptive effects of spinally administered neuropeptide Y in the rat: systematic studies on structure-activity relationship. J. Pharmac. Exp. Ther. 258: 243-248. Iadorola M. J., Tang J., Costa E. and Yang H.-Y. T. (1986) Analgesic activity and release of [Met’lenkephalin-Arg6Gly’-Leu’ from rat spinal cord in vivo. Eur. J. Pharmac. 1%: 39-48.

Inturrisi E. E., Umans J. G., Wolff D., Stern A. S., Lewis R. V., Stein S. and Udenfriend S. (1980) Analgesic activity of the naturally occurring heptapeptide [Met]-enkephalin-Arg6Phe’. Proc. Natn. Acad. Sci. U.S.A. 77: 5512-5514. Larson A. S., Vaught J. L. and Takemori A. E. (1980) The potentiation of spinal analgesia by leucine enkephalin. Eur. J. Pharmac. 61: 381-383.

Liston D., Datey G., Rossier J., Verbanck J.-J. and Vanderhaeghem J.-J. (1984) Processing of proenkephalin is tissue specific. Science 225: 734-737. Loomis C. W., Jhamandas K., Milne B. and Cervenko F. (1987a) Monoamine and opioid interactions in spinal analgesia and tolerance. Pharmac. Biochem. Behav. 26: 445-451. Loomis C. W., Milne B. and Cervenko F. W. (1987b) Determination of cross tolerance in rat spinal cord using intrathecal infusion via sequential mini-osmotic pumps. Pharmac. Biothem. Behav. 26: 131-139.

Lord J. A. H., Waterfleld A. A., Hughes J. and Kosterlitz H. W. (1977) Endogenous opioid peptides: multiple agonists and receptors. Nature 267: 49-99. Malhotra R. K., Wakade T. D. and Wakade A. R. (1988) Comparison of secretion of catecholamines from the rat adrenal medulla during continuous exposure to nicotine, muscarine or excess K. Neuroscience 26: 313-320. Porreca F., Jiang Q. and Tallarida R. J. (1990) Modulation of morphine antinociception by peripheral [LeuSlenkephalin: a synergistic interaction. Eur. J. Pharmac. 179: 463-468. Ramabadran K. and Bansinath M. (1986) A critical analysis of experimental evaluation of nociceptive reactions in animals. Pharm. Res. 3: 263-270. Russell R. D., Leslie J. B., Su Y.-F., Watkins W. D. and Chang K.-J. (1987) Continuous intrathecal opioid analgesia: tolerance and cross-tolerance of mu and delta spinal opioid receptors. J. Pharmac. Exp. Ther. 240: 150-158. Sagen J. and Kemmler J. (1989) Increased levels of metenkephalin-like immunoreactivity in the spinal cord CSF of rats with adrenal medullary transplants. Brain Res. 502: l-10. Sagen J., Kemmler J. E. and Wang H. (1991) Adrenal medullary transplants increase spinal cord cerebrospinal fluid catecholamine levels and reduce pain sensitivity. J. Neurochem. 56: 623-627.

Sagen J. and Pappas G. D. (1987) Morphological and functional correlates of chromaffin cell transplants in CNS pain modulatory regions. In: Ann. N.Y. Acad. Sci., Cell and Tissue Transplantation in the Adult Brain (Azmitia E. C. and Bjorklund A., Eds), Vol. 495, pp. 306333. New York Academy of Sciences, N.Y. Sagen J., Pappas G. D. and Perlow M. J. (1986a) Adrenal medullary tissue transplants in the rat spinal cord reduce pain sensitivity. Bruin Res. 384: 189-194. Sagen J., Pappas G. D. and Pollard H. B. (1986b) Analgesia induced by isolated bovine chromaffin cells implanted in rat spinal cord. Proc. Natn. Acad. Sci. U.S.A. 83: 7522-7526..

692

H. WANG and J. SAGEN

Sagen J., Wang H. and Pappas G. D. (1990) Adrenal medullary implants in rat spinal cord reduce nociception in a chronic pain model. Pain 42: 69-79. Sagen J., Wang H., Tresco P. A. and Aebischer P. (1994) Transplants of immunologically isolated xenogeneic chromaffin cells provide a long-term source of pain-reducing neuroactive substances. J. Neurosci. 13: 2415-2423. Sanchez-Blazquez P. and Garzon J. (1993) N-acetyl /3-Endorphin-(l-31) and Substance P regulate the supraspinal antinociception mediated by mu opioid and alpha-2 adrenoceptors but not by delta opioid receptors in the mouse. J. Pharm. Exp. Ther. 265: 835-843. Sahley T. L. and Berntson G. G. (1979) Antinociceptive effects of central and systemic administrations of nicotine in the rat. Psychopharmacology 65: 279-283.

Schiavone M. T. and Kirpekar S. M. (1982) Inactivation of secretory responses to potassium and nicotine in the cat adrenal medulla. J. Pharmac. Exp. Ther. 223: 743-749. Sherman S. E., Loomis C. W., Milne B. and Cervenko F. W. (1988) Intrathecal oxymetazoline produces analgesia via spinal cr-adrenoceptors and potentiates spinal morphine. Eur. J. Pharmac. 148: 371-380. Stern A. S., Lewis R. V., Kimura S., Rossier J., Stein S. and Udenfriend S. (1980) Opioid hexapeptides and heptapeptides in adrenal medulla and brain: possible implications on the biosynthesis of enkephalins. Archs Biochem. Biophys. 205: 606-613.

Stevens C. W., Monasky M. S. and Yaksh T. L. (1988) Spinal infusion of opiate and alpha-2 agonists in rats: tolerance and cross-tolerance studies. J. Pharmac. Exp. Ther. 244: 63-70. Stevens K. E., Leslie F. M., Evans C. J., Belluzzi J. D. and Stein L. (1988) BAM-18: analgesia, hyperalgesia, and locomotor effects. Neuropeptides 12: 21-27. Tallarida R. J. and Murray R. B. (1987) Manual of Pharmacologic Calculations with Computer Programs. Springer-Verlag, N.Y. Tripathi H. L., Martin B. R. and Aceto M. D. (1982) Nicotineinduced antinociception in rats and mice: correlation with nicotine brain levels. J. Pharmac. Exp. Ther. 221: 91-96. Tseng L.-F. (1982) Tolerance and cross-tolerance to morphine after chronic spinal D-Ala*-D-LeuS-enkephalin infusion. Life Sci. 31: 987-992.

Tseng L.-F. (1983) Partial cross tolerance to D-Ala’-D-Leu5enkephalin after chronic spinal morphine infusion. Life Sci. 32: 25452550. Tyler C. B. and Advokat C. (1986) Investigation of “cross-tolerance” between systemic and intrathecal morphine in rats. Physiol. Behav. 27: 27-32.

Wang H. and Sagen J. (1989) Tolerance studies on pain reduction by adrenal medullary implants in the spinal cord subarachnoid space. Sot. Neurosci. Abstr. 15: 1243. Wang H. and Sagen J. (1993) Role of opiate receptor subtypes in mediating antinociception by adrenal medullary transplants in the rat spinal subarachnoid space. Sot. Neurosci. Abstr. 19: 1410. Wilcox G. L., Carlsson K., Jochim A. and Jurna I. (1987) Mutual potentiation of antinociceptive effects of morphine and clonidine on motor and sensory responses in rat spinal cord. Brain Res. 405: 84-93. Yaksh T. L. (1984) Multiple spinal opiate receptor systems in analgesia. In: Advances in Pain Research and Therapy (Kruger L. and Liebeskind J. C., Eds), Vol. 6, pp. 197-215. Raven Press, New York. Yaksh T. L. (1987) Spinal opiates: a review of their effect on spinal function with emphasis on pain processing. Acta Anaesthesiol. Stand. 31 Suppl.: 25-37. Yaksh T. L., Howe J. R. and Harty G. J. (1984) Pharmacology of spinal pain modulatory systems. Adv. Pain Res. Ther. 7: 57-70.

Yaksh T. L., Kohl R. L. and Rudy T. A. (1977) Induction of tolerance and withdrawal in rats receiving morphine in the spinal subarachnoid space. Eur. J. Pharmac. 42: 275-284.

Yaksh T. L. and Reddy S. V. R. (1981) Studies in the primate on the analgetic effects associated with intrathecal actions of opiates, alpha-adrenergic agonists, and baclofen. Anesthesiology 54: 451467.

Zenz M., Piepenbrock S. and Tryba M. (1985) Epidural opiates: long-term experiences in cancer pain. Klin. Wochen schr. 63: 225-229.

Zhang A.-Z. and Pasternak G. W. (1981) Ontogeny of opioid pharmacology and receptors: high and low affinity site differences. Eur. J. Pharmac. 73: 2940.