Brain Research, 384 (1986) 189-194 189 Elsevier

BRE 21813

Adrenal medullary tissue transplants in the rat spinal cord reduce pain sensitivity

JACQUELINE SAGEN 1, GEORGE D. PAPPAS t and MARK J. PERLOW 2

Departments of 1Anatomy and 2Neurology, University of Illinois at Chicago, Chicago, IL 60612 (U.S.A.)

(Accepted 17 June 1986)

Key words: Neural transplant - - Adrenal medulla - - Chromaffin cell - - Analgesia - - Nociception - - Opioid peptide

Adrenal chromaffin cells contain and release several neuroactive substances which induce analgesia when injected directly into the spinal cord (e.g. opioid peptides and catecholamines). Furthermore, the release of these substances can be induced by nicotine. In or- der to determine whether adrenal medullary tissue transplanted to the spinal cord can produce alterations in pain sensitivity, pieces of dissected rat adrenal medulla were placed in the subarachnoid space of rat spinal cords. Stimulation by a low dose of nicotine induced potent analgesia in animals with adrenal medullary transplants, but not in animals with control transplants. Furthermore, this analge- sia was reversed to pre-nicotine levels by the opiate antagonist naloxone. Thus adrenal medullary transplants in the spinal cord may provide a permanent and locally available source of opioid peptides for the relief of intractable pain.

The ability to successfully transplant neural tissue into the central nervous system (CNS) of adult hosts for prolonged periods of t ime is fairly well estab- lished 2'4'2°. This approach is potentially clinically use-

ful for the replacement of damaged neuronal circuit- ry. Improvements in reproductive behavior 7, cogni- tion 6 and motor behavior t4 in lesioned animals have

recently been repor ted following the transplantation of appropriate fetal neuronal tissue.

Recent studies in our and other laboratories have demonstra ted that adult adrenal medullary tissue transplanted into the CNS also survives and has the potential to restore functional deficits in lesioned ani- mals 5'15. We have been interested in the control of

pain sensitivity in intact, non-lesioned animals. Ad- renal chromaffin cells are ideal candidates for trans- plantation since they contain and release substances known to alter pain sensitivity when injected into the spinal cord (e.g. Met- and Leu-enkephal in, norepi- nephrine, epinephrine9,13,22,25). Moreover , the re-

lease of these substances can be induced by pharma- cological agents such as nicotine.

The presence of enkephalin-containing neurons and opiate receptors in high densities in the substan- tia gelatinosa of the spinal cord 1's'11 and the resultant

analgesia observed following local injection of opiates into the spinal cord 24 have suggested a role

for opioid peptides in modulat ing the central trans- mission of nociceptive information. In addition, cate-

cholamines also appear to be important in modulat- ing pain sensitivity in the spinal cord since the injec- tion of noradrenergic agonists into the subarachnoid space of the spinal cord produces analgesia 1°,t6, while

the injection of noradrenergic antagonists produces increased sensitivity to noxious stimuli 19. Chromaffin cell transplants would provide a local and readily available source of these agents in the spinal cord. Results of the present study demonstra te that it is

possible to reduce pain sensitivity following the transplantation of adrenal medullary tissue into the spinal cord. A prel iminary account of this work was reported previously is.

Baseline pain sensitivity was determined in female Sprague-Dawley-der ived rats weighing 250-300 g, using 3 analgesiometric tests in succession: the tail flick test 3, the paw pinch test and the hot plate test 23.

To elicit the tail flick response, a focused beam of high intensity light is applied to the dorsal surface of

the rat 's tail. The time interval between the onset of the s t imulusand the tail flick response is measured at

Correspondence: G.D. Pappas, Department of Anatomy, University of Illinois at Chicago, 808 South Wood St., Chicago, IL, 60612, U.S.A.

0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

190

3 regions of the rat 's tail, the average of which is de-

fined as the 'tail flick latency' . To prevent tissue da-

mage in the absence of a response, the stimulus is ter-

minated at 14 s and the tail flick latency assigned a

value of 14. The hot plate response is de te rmined by

placing the rats on a 55 °C copper plate enclosed in a

plexiglass cylinder. The intervals be tween p lacement

on the hot plate and the response of ei ther licking the

hind paws or jumping off the plate is defined as the

'hot plate latency' . In the absence of a response, the

animal is removed after 40 s and assigned a hot plate

latency of 40. The paw pinch response is elicited by a

Ugo-Basi le Ana lgesy-Meter which applies pressure

at a constant rate of 64 g/s. The force is appl ied to the

ventral surface of both hind paws sequential ly until

the animal reacts by a withdrawal response.

Baseline pain sensitivity and pain sensitivity fol-

lowing a low dose of nicotine (0.1 mg/kg, s.c.) were

de termined pr ior to surgical procedures . Adrena l tis-

sue for t ransplantat ion was obta ined from female

Sprague -Dawley-de r ived rats of the same group as

the host animals. Adrena l medul lary tissue was dis-

sected from cortical tissue, cut into small pieces (less

than 0.5 mm3), and incubated in 2.5 S nerve growth

factor (0.1 #g/ml) in Hank ' s buffer containing 1

mg/ml bovine serum albumin for 20 min. Tissue

from one adrenal medul la was t ransplanted in each

animal. Control animals received an equal volume of

ei ther heat-ki l led adrenal medul lary tissue or sciatic

nerve tissue. Under pentobarb i ta l anesthesia (30

mg/kg, i .p.) , a laminectomy was per formed to expose

a 2 - 3 - m m segment of the lumbar enlargement . Un-

der a dissecting microscope, a small incision was

made in the dura and pieces of adrenal medul la were

placed in the subarachnoid space and pushed under

the dura to keep them in place. The skin was closed

with wound clips and the animals re turned to their

cages for observat ion. Animals exhibit ing motor ab-

normali t ies following surgical procedures were dis-

carded from the study.

Pain sensitivity was de termined at several intervals

following t ransplantat ion, but the 8-week t ime point

is repor ted here since morphological studies have

shown the grafts are well es tabl ished at this t ime. Fol-

lowing determinat ion of pain sensitivity in trans-

p lanted animals, response to nicotine s t imulat ion (0.1 mg/kg, s.c.) was measured at 2, 10, 20 and 30

rain following the injection. Results of this study are

shown in Fig. 1. Animals receiving control trans-

plants of either killed adrenal tissue or sciatic nerve

tissue were pooled since there was no difference in

their responses. Pain sensitivity in pre- implanted ani-

T A I L h L I r . . K L A T E N C Y ( s e c )

10

HOT P L A T E L A T E N E Y ( s e c ) B

2 5

2 0

15

10

5 i , , , , i . . . . i , , , , i

PAW P I N C H T H R E S H O L O C

2 0

1 5

1 0

5 i , , , , i , , , , i , , , , i

0 • I 0 20 30

TIME (min)

Fig. 1. Effect of spinal cord adrenal medullary transplants on pain sensitivity. The ordinate is the threshold for response to noxious stimuli as assessed by the tail flick test (A), hot plate test (B) or paw pinch test (C). Each point represents the mean + S.E.M. The abscissa is the time course of responses to nox- ious stimuli following nicotine stimulation. Time 0 indicates the pre-injection values. The arrowhead indicates the point at which nicotine (0.1 mg/kg, s.c.) was injected. Symbols: circles, animals with adrenal medullary transplants in the spinal cord (n = 12); squares, animals with control transplants in the spinal cord (n = 10). Comparisons between the two groups using two- way ANOVA indicated that the induction of analgesia was sta- tistically significant for all 3 tests (P < 0.01, tail flick and hot plate tests; P < 0.05. paw pinch test).

mals (not shown) was 3.2 _+ 0.2, 7.7 + 0.8 and 9.5 + 0.5 s, for the tail flick, hot plate and paw pinch tests, respectively. The injection of nicotine had no effect on the pain threshold of these animals prior to im- plantation. Similarly, the injection of nicotine did not alter pain responsiveness in control transplant ani- mals (Fig. 1). In contrast, this dose of nicotine pro- duced potent analgesia in animals with spinal cord adrenal medullary transplants. The reduction in pain responsiveness was observed for all 3 analgesiome- tric tests (Fig. 1). This analgesia was apparent 2 min following nicotine stimulation, and pain threshold re- mained elevated for 10-20 min following the injec- tion, tending toward baseline by 30 min.

Since nicotine is well known to induce the release of chromaffin cell granule contents, it is likely that these results are due to the local release of these sub- stances into the spinal cord subarachnoid space. In order to determine whether the observed analgesia was due to the stimulated release of opioid peptides from the adrenal medullary transplants, a second group of animals received identical transplants. Im- mediately following the induction of analgesia by nic- otine (0.1 mg/kg, s.c.), animals received an injection of either opiate antagonist naloxone (2 mg/kg, s.c.) or saline vehicle. Results of this study are shown in Fig. 2. The injection of naloxone following nicotine- stimulated analgesia dramatically reversed this anal- gesia. Pain threshold was reversed to baseline levels on all 3 tests at 10 min and remained there for the du- ration of testing. In contrast, pain threshold re- mained elevated in animals receiving saline vehicle injections.

To control for the tendency of the elevation in pain sensitivity to gradually decrease toward pre-nicotine baseline levels over time, some transplanted animals received naloxone injections prior to nicotine stimu- lation. The induction of analgesia by nicotine was completely blocked in these animals. Together, these results suggest that the stimulated release of opioid peptides from chromaffin cells transplanted into the spinal cord is at least partially responsible for the in- duction of analgesia. Determination of the contribu- tion of catecholamines to this effect is currently in progress. Since in situ rat adrenals contain relatively low levels of enkephalins, it is possible that increased enkephalin synthesis is induced in chromaffin cells when transplanted to the CNS environment. This

191

TAIL FLICK LATENCY (sec)

8

4

HOT PLATE LATENCY (sec) 15

10

A

, , , , i i , , , i

B

5i i , , , , i , , , , i , , , , i

PAW PINCH THRESHOLO C 20

15

I0

10 20 30

TIME (min)

Fig. 2. Effect of naloxone on the analgesia induced by spinal cord adrenal medullary transplants. The ordinate is the thresh- old for response to noxious stimuli as assessed by the tail flick test (A), hot plate test (B) or paw pinch test (C). Each point represents the mean + S.E.M. The abscissa is the time course of responses to noxious stimuli following drug injections. Time 0 indicates the pre-injection values. The closed arrowhead indi- cates the point at which nicotine (0.1 mg/kg, s.c.) was injected. Following the induction of analgesia, animals received either naloxone (2 mg/kg, s.c.) or saline vehicle, indicated at the open arrowhead. Symbols: circles, animals with spinal cord adrenal medullary transplants receiving naloxone following the induc- tion of analgesia (n = 9); squares, animals with spinal cord ad- renal medullary transplants receiving saline following the in- duction of analgesia (n = 9). Comparisons between the two groups using two-way ANOVA indicated that the reversal of the analgesia by naloxone was statistically significant for all 3 tests (P < 0.01).

possibility is supported by studies showing increased enkephalin levels in rat adrenals following denerva- tion and in human chromaffin cells in culture 12'2t.

In order to determine the underlying morphologi- cal changes responsible for the observed alterations in pain sensitivity, animals were prepared for elec- tron microscopy following termination of behavioral testing. Animals were deeply anesthetized and per- fused via the aorta with saline followed by buffered mixed aldehydes. The spinal cords were removed and routinely processed for electron microscopy. The transplanted tissue was readily identified under a dissecting microscope and Toluidine blue-stained semi-thin sections revealed that the transplants were healthy and contained numerous chromaffin cells (Fig. 3A, B). Ultrastructural observations revealed that the chromaffin cells in the transplants contained many chromaffin granules, primarily of the norepi- nephrine-containing type in that they are oval and have dense cores (Fig. 3B, D), in contrast to the epi- nephrine type found predominantly in the intact ad- renal which are round and have a more even density.

The capillaries in the transplants appear to be fenes- trated (Fig. 3C), in contrast to those of the host CNS, providing a potential for leakiness in the blood-brain barrier 17. Interestingly, there does not appear to be

extensive interaction between the graft tissue and the host CNS, although finger-like projections contain- ing chromaffin granules can occasionally be seen pro- truding from chromaffin cells in the graft (Fig. 3E). Thus it does not appear that synaptic relationships between the host and graft tissue are a necessary pre- requisite for behavioral alterations when the tissue transplants are placed under the dura and not directly into the CNS parenchyma. Rather, it is likely that the observed alterations in pain sensitivity are due to the humoral release of pharmacologically active sub- stances into the subarachnoid space of the spinal

193

cord.

In conclusion, results of these studies indicate that

adrenal chromaffin cells thrive when transplanted to the subarachnoid space of the spinal cord. Moreover, these transplanted cells produce potent reduction in pain responsiveness when stimulated by nicotine. The observation that this analgesia is reversed by nal- oxone suggests that it may be induced by the stimu- lated release of opioid peptides from granules of transplanted chromaffin cells. Thus, the transplanta- tion of adrenal medullary tissue may provide a per- manent and readily available local source of opioid peptides for the relief of intractable pain. One aspect that requires further study is the question of toler-

ance to repeated release of opioid peptides from transplanted chromaffin cells. An interesting obser- vation by Yaksh and Reddy 24 is that while there is sig- nificant tolerance to analgesic potency with repeated

intrathecal morphine injections, the concurrent in- jection of lower doses of morphine and a noradrener-

gic agonist circumvents this decrement in analgesic potency. Since chromaffin cells release both opioid peptides and catecholamines, they may provide an ideal combination for avoiding the development of tolerance. Preliminary work in our laboratory sug-

gests that tolerance does not develop, since analgesia can be repeatedly induced at daily intervals in ani- mals with spinal cord adrenal medullary implants.

We acknowledge the generous support and advice of Dr. Herbert K. Proudfit in lending us his analge- siometric equipment. We gratefully acknowledge the electron microscope facility of the Research Re- sources Center, University of Illinois at Chicago, for the use of their equipment. This work was supported, in part, by a National Research Service Award (NIH 1-F32NS07630) and NIH GM 37326 Research Grant.

Fig. 3. A: light micrograph of a section from a portion of the dorsal lumbar spinal cord. A piece of adrenal medullary tissue transplant can be found in the subdural space. Dense chromaffin cells can be readily identified in the graft (arrowheads). D, dura; sp c, spinal cord. B: electron micrograph of a portion of adrenal medullary tissue that was transplanted for 8 weeks in the spinal cord. The graft is made up primarily of compact granulated chromaffin cells. Chromaffin granules (in the lower left at large asterisk) are shown enlarged in D. Note that portions of the blood vessel endothelial cells are attenuated and fenestrated. (See C which is an enlargement of the en- dothelial process at the small asterisk.) C, collagen; L, lumen of blood vessel; m, mitochondria; n, nucleus of chromaffin cell. C: dia- phragms at arrowheads are seen in the fenestrae of the endothelial process, enlarged from B. D: the granules enlarged from B, are of the norepinephrine type, in contrast to the predominant epinephrine type found in the rat adrenal medulla in situ. E: electron micro- graph of a portion of a chromaffin cell in the subarachnoid space of the dorsal spinal cord where some short blunt processes are present 8 weeks following transplantation.

194

1 Atweh, S.F. and Kuhar, M.J., Autoradiographic localiza- tion of opiate-receptors in rat brain. II. The brainstem, Brain Research, 129 (1977) 1-12.

2 Bjorklund, A. and Stenevi, U., Neural Grafting in Mamma- lian CNS, Elsevier, Amsterdam, 1985, 709 pp.

3 D'Amour, F.E. and Smith, D.L., A method for determin- ing loss of pain sensation, J. Pharmacol. Exp. Ther., 72 (1941)74-79.

4 Das, G.D. and Wallace, R.B., Neural Transplantation and Regeneration, Springer, New York, 1986, 330 pp.

5 Freed, W.J., Morihisa, J.M., Spoor, E., Hoffer, B.J., O1- son, L., Seiger, A. and Wyatt, R.J., Transplanted adrenal chromaffin cells in rat brain reduce lesion-induced rotation- al behavior, Nature (London), 292 (1981) 351-352.

6 Gage, F.H., Bjorklund, A., Stenevi, U., Dunnett, S.B. and Kelly, P.A.T., Intrahippocampal septal grafts ameliorate learning impairments in aged rats, Science, 225 (1984) 533-536.

7 Gibson, M.J., Kreiger, D.T., Charlton, H.M., Zimmer- man, E.A., Silverman, A.J. and Perlow, M.J., Mating and pregnancy can occur in genetically hypogonadal mice with preoptic area brain grafts, Science, 225 (1984) 949-951.

8 Glazer, E.J. and Basbaum, A.I., Immunohistochemical lo- calization of leucine-enkephalin in the spinal cord of the cat: enkephalin-containing marginal neurons and pain modulation, J. Comp. Neurol., 196 (1981) 377-389.

9 Kondo, H., Immunohistoehemical analysis of the localiza- tion of neuropeptides in the adrenal gland, Arch. Histol. Jap., 48 (1985) 453-481.

10 Kuraishi, Y., Harada, Y. and Takagi, H., Noradrenaline regulation of pain-transmission in the spinal cord mediated by alpha-adrenoceptors, Brain Research, 174 (1979) 333-336.

11 LaMotte, C., Pert, C.B. and Snyder, S.H., Opiate receptor binding in primate spinal cord. Distribution and changes af- ter dorsal root section, Brain Research, 112 (1976) 407-412.

12 Lewis, R.V., Stern, A.S., Kilpatrick, D.L., Gerber, L.D., Rossier, J., Stein, S. and Udenfriend, S., Marked increases in large enkephalin-containing polypeptides in the rat adre- nal gland following denervation, J. Neurosci., 1 (1981) 80-82.

13 Livett, B.G., Dean, D.M., Whelan, L.G., Udenfriend, S. and Rossier, J., Co-release of enkephalin and catechol-

amines from cultured adrenal chromaffin cells, Nature (London), 289 (1981) 317-319.

14 Perlow, M.J., Freed, W.J., Hoffer, B.J., Seiger, A., Ol- son, L. and Wyatt, R.J., Brain grafts reduce motor abnor- malities produced by destruction of the nigrostriatal dopa- mine system: behavioral histoehemical evidence, Science, 204 (1979) 643-647.

15 Perlow, M.J., Kumakura, K. and Guidotti, A., Prolonged survival of bovine adrenal chromaffin cells in rat cerebral ventricles, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 5278-5281.

16 Reddy, S.V.R., Maderdrut, J.L. and Yaksh, T.L., Spinal cord pharmacology of adrenergic agonist-mediated antino- ciception, J. Pharmacol. Exp. Ther., 213 (1980) 525-533.

17 Rosenstein, J.M., The blood brain barrier is permanently altered by transplants of adrenal medulla, Soc. Neurosci. Abstr., 15 (1985) 840.

18 Sagen, J., Pappas, G.D. and Perlow, M.J., Alterations in pain sensitivity following the transplantation of adrenal medullary tissue into the spinal cord, Soc. Neurosci. Abstr., 11 (1985) 615.

19 Sagen, J. and Proudfit, H.K., Effect of intrathecally admin- istered noradrenergic antagonists on nociception in the rat, Brain Research, 310 (1984) 295-301.

20 Sladek, J. and Gash, D., Neural Transplants, Development and Function, Plenum Press, New York, 1984.

21 Tischler, A.S., DeLeUis, R.A., Biales, B., Nunnemacher, G., Carabba, V. and Wolfe, H., Nerve growth factor-in- duced neurite outgrowth from normal human chromaffin cells, Lab. Invest., 43 (1980) 399-409.

22 Wilson, S.P., Chang, K.-J. and Viveros, O.H., Proportio- nal secretion of opioid peptides and catecholamines from adrenal chromaffin cells in culture, J. Neurosci., 2 (1982) 1150-1156.

23 Woolfe, G. and MacDonald, A.D., The evaluation of the analgesic action of pethidine hydrochloride (Demerol), J. Pharmacol. Exp. Ther., 80 (1944)300-307.

24 Yaksh, T.L. and Reddy, S.V.R., Studies in the primate on the analgetic effects associated with intrathecal actions of opiates, alpha-adrenergic agonists and baclofen, Anesthe- siology, 54 (1981) 451-467.

25 Yang, H.-T., Hexum, T. and Costa, E., Opioid peptides in adrenal gland, Life Sci., 27 (1980) 1119-1125.