Inducing analgesia by implantation of cells releasing neuroactive substances

A method of inducing analgesia or reducing pain is described. The method involves implanting in the central nervous system of a host susceptible to pain, living material capable of releasing effective amount of analgesic substance when interacted with a stimulus which induces said material to release analgesic amount of said substance. The procedure described herein reduces sensitivity to intractable pain.

BACKGROUND OF THE INVENTION 
1. Technical Field 
The present invention is related to producing analgesia or reducing pain by 
implantation of cellular material in the central nervous system (CNS) of a 
host susceptible to pain sensation. More particularly, the present 
invention is related to the implantation of isolated chromaffin cells or 
adrenal medullary tissue in the brain stem or spinal cord for producing 
analgesia when the implanted tissue or cell is induced to release 
nociceptor interacting substances. 
2. State of the Art 
Reports of successful implantation of nonhomologous neural tissue to the 
central nervous system without immunological rejection have been known and 
these reports 
Holman & Stern, Chartered Folio P49667 
have created interest in the potential of such transplants for restoring 
functional deficits. Improvements in reproductive behavior, cognition and 
motor behavior in lesioned animals have been reported following the 
implantation of appropriate fetal neuronal tissue. Implants of adrenal 
chromaffin cells have been shown to survive for long periods of time when 
transplanted into the central nervous system (Perlow et al., 1980, Proc. 
Natl. Acad. Sci. 77:5278-5281). Recently, adrenal medullary homografts 
have been implanted in the striatum of human Parkinson patients with 
encouraging results (Backlund et al., 1985, J. Neurosurg. 62:169-173). 
However, modulation of pain sensitivity by implants of living cells or 
tissue in the CNS has not heretofore been attempted. Since pain is not 
necessarily the result of damaged neuronal tissue, it is essential that 
the function of neural implants in intact, non-lesioned systems be 
assessed when determining pain sensitivity. 
SUMMARY OF INVENTION 
It is, therefore, an object of the present invention to establish the 
efficacy for pain relief of CNS implants of such cells or tissue which 
release neuroactive substances when induced with suitable stimulus. 
It is a further object of the present invention to provide a method of 
producing analgesia or reducing pain by implanting in the central nervous 
system such cells or tissues which release nociceptor reacting substances 
when induced with a suitable stimulus. 
Other objects and advantages will become evident as the detailed 
description of the invention proceeds.

DETAILED DESCRIPTION OF INVENTION 
The above and other objects and advantages of the present invention are 
achieved by a novel method of relieving pain comprising implanting in the 
CNS of a host susceptible to pain, living cells or tissues capable of 
releasing effective amount of analgesic substances when interacted with a 
stimulus which induces said cells or tissues to release analgesic 
substances including opioid peptides and catecholamines. Of course, any 
suitable type of living material can be employed so long as such material 
is transplantable in the CNS without rejection and has the inherent 
property of locally or systemically releasing in the tissue, body or body 
fluid analgesic substances under appropriate conditions. Preferred 
examples of such material are isolated, substantially homogeneously 
purified bovine chromaffin cells and adrenal medullary tissues. 
Implantation of such material can be done at any suitable site in the CNS 
where nociceptors are accessibly present. Typical examples of such sites 
are spinal cord, and the brainstem, particularly the dorsal horn, the 
subarachnoid space of the lumbar region, substantia gelatinosa, the 
periaqueductal gray, the nucleus raphe magnus, the nucleus reticularis 
gigantocellularis and the like. Implantation in one or more of such 
regions would provide a permanent and continuous local source of 
neuroactive substances, thus providing a means for permanent relief of 
pain or for reducing sensitivity to intractable pain. Moreover, the level 
of pain relief can be easily controlled or modulated pharamacologically by 
using any agent which stimulates receptors on the implanted cells, such as 
nicotine or muscarinic agonists and the like. Analgesia thus induced can 
also be conveniently manipulated or reversed by such antagonists as the 
opiate antagonist naloxone, adrenergic antagonist phentolamine, ganglionic 
antagonist mecamylamine, and the like. Stimulation or modulation of 
implanted cells can, of course, also be achieved by other means such as 
delivering pulses of electrical current through micro-electrodes and the 
like. 
Unless specifically defined otherwise, all scientific or technical terms 
used herein have the same meaning as generally understood in the art to 
which the present invention belongs and all publications mentioned 
hereunder are incorporated herein by reference. Although any similar or 
equivalent methods and materials can be conveniently adopted in the 
practice or testing of the invention disclosed herein, the preferred 
methods and mateials are now described. 
MATERIALS AND METHODS 
In all of the following studies, male Sprague-Dawley derived rats weighing 
300-500 grams served as hosts. Pain sensitivity was measured in these 
animals using three standard analgesiometric tests sequentially: the tail 
flick test, the paw pinch test, and the hot plate test. 
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 stimulus and the tail flick response is measured 
at three regions of the tail, the average of which is defined as the "tail 
flick latency". To prevent tissue damage in the absence of a response, the 
stimulus is terminated at 14 seconds and the tail flick latency is 
assigned a value of 14. The paw pinch response is elicited by a 
commercially available apparatus (Ugo-Basile) which applies pressure at a 
constant rate of 64 grams/sec. The force is applied to the ventral surface 
of both hind paws sequentially until the animal reacts by a withdrawal 
response. The hot plate response is determined by placing the rat on a 
55.degree. C. copper plate enclosed in a plexiglass cylinder. The interval 
between placement on the hot plate is defined as the "hot plate latency". 
In the absence of a response, the animal is removed after 40 seconds and 
assigned a hot plate latency of 40. Thus, both thermal and mechanical pain 
stimuli were employed, as well as both reflexive and integrated pain 
response were monitored. In all of the following studies, animals were 
initially screened for baseline pain sensitivities and pain sensitivities 
following a low dose of nicotine (0.1 mg/kg, subcutaneously). 
EXAMPLE 1 
The isolated chromaffin cells were obtained from the adrenal glands of 
steers or cows as described by Pollard et al., J. Biol. Chem. 
259:1114-1121 (1984). The resultant preparation from 7-9 glands contains 
about 0.5-1.0.times.10.sup.9 chromaffin cells and is substantially pure, 
that is essentially free of other cell types. Suspensions of primary 
cultures of bovine chromaffin cells were obtained in air-tight culture 
media at 4.degree. C. the day after preparation. Of course, it should be 
understood that bovine source for chromaffin cells is used simply because 
of convenience and easy availability. But, any other source which is 
deemed suitable can be equally well used, the source per se of the tissue 
or cells not being a critical feature of the invention. 
For implantation, the cells were concentrated by centrifugation and 
resuspended in small volumes of Hank's buffer containing 0.1 .mu.g/ml 2.5 
S nerve growth factor and kept on ice until they were placed in the rat 
spinal cords. The cells were injected via an intrathecal catheter 
according to a modification of the technique of Yaksh and Rudy, Physiol. 
Behav. 27:1031-1036(1976). Under ether anesthesia, a small incision was 
made in the dura overlying the atlanto-occipital junction, a catheter made 
of polyethylene (PE 10) tubing was threaded through the incision into the 
subarachnoid space and down the spinal cord to the level of the lumbar 
enlargement. Cell suspensions were injected through the catheter in 15 
.mu.l volumes over 20-30 seconds, followed by a 10 .mu.l flush with Hank's 
buffer. Each animal received approximately 100,000 cells (counted in a 
hemocytometer). Cell viability was determined at the end of the surgical 
procedures by trypan blue exclusion to be 80-90%. Control animals received 
equal volumes of either heat-skilled cells or only Hank's buffer 
containing nerve growth factor. 
Animals which exhibited motor abnormalities following surgical procedures 
were discarded from the study. The remaining animals were returned to 
their cages and allowed free access to food and water. They were tested 
for pain sensitivities according to the protocols described hereunder. 
Test 1 
Initially, animals were tested 6-8 weeks following cell implantation, since 
this was determined to be sufficient for establishment of behavioral 
responses. Pain sensitivity was assessed for implanted and control animals 
by the three analgesiometric tests listed above. The animals then received 
an injection of nicotine (0.1 mg/kg, s.c.), and were tested again 2, 10, 
20, and 30 minutes later. 
Test 2 
In order to determine the potential for long term changes in pain 
sensitivity, another group of animals with identical implants were tested 
for pain responsiveness before and after nicotine at several time 
intervals following the implantation procedures. Rats were tested at 1 
day, 1 week, 2 weeks, 4 weeks, 8 weeks, and 16 weeks after receiving cell 
or control implants. 
Test 3 
To determine the sensitivity of chromaffin cell implants to nicotine, 
another group of implanted animals received several doses of nicotine at 
weekly intervals on a rotating dose schedule. The doses of nicotine used 
were 0.05 mg/kg, 0.1 mg/kg, and 0.2 mg/kg. 
Test 4 
Since the analgesia induced by stimulation of chromaffin cells may be due 
to the release of neuroactive substances from chromaffin cell granules, it 
was important to determine the contribution of catecholamines and opioid 
peptides to this response. Rats with spinal cord bovine chromaffin cell 
implants received an injection of either opiate antagonist naloxone (2 
mg/kg, s.c.), alpha-adrenergic antagonist phentolamine (10 mg/kg, s.c.), 
or saline vehicle 5 minutes before the nicotine injection. These 
antagonist doses were chosen since they do not produce any alterations in 
pain sensitivity (Jensen et al., Eur. J. Pharmacol. 86:65-70, 1983). 
Statistical analysis was done using two-way analysis of variance (ANOVA) 
and the Newman-Keuls test for multiple post-hoc comparisons (Keppel et 
al., Design and Analysis: A Researcher's Handbook, 1973). 
The results indicated that prior to the implantation of chromaffin cells, 
nicotine (0.1 mg/kg) did not produce any alterations in pain sensitivity 
as assessed by the tail flick, paw pinch, or hot plate tests. In contrast, 
the injection of nicotine induced potent analgesia in animals with spinal 
cord chromaffin cell implants (P&lt;0.01 for all three tests). The results 
are shown in FIG. 1. The peak increase in pain threshold was at 2 minutes 
following the nicotine injection. Both tail flick latency and paw pinch 
threshold remained elevated for 20 minutes, tending toward baseline levels 
by 30 minutes, while hot plate latencies returned to baselines by 20 
minutes. The injection of nicotine had not significant effect on pain 
sensitivity in animals with control implants. 
The ability of nicotine to induce analgesia in implanted animals was tested 
at several intervals over a 16 week period. Results are summarized in FIG. 
3. Since this dose of nicotine did not significantly alter pain 
sensitivity at any time in control animals (P&gt;0.05), the data for these 
animals has not been included herein. Analgesia induced by nicotine 
stimulation could be observed as early as one day following cell 
implantation. However, at this time, the difference between the pre- and 
post-nicotine response latencies were smaller than at other time points, 
particularly for the tail flick and paw pinch tests. An explanation for 
this is that the baseline pain sensitivities (pre-nicotine) were higher at 
one day following cell implantation than at other times during the study. 
Compared to the pre-implantation pain sensitivities, tail flick latency 
was elevated from 3.2+/-0.4 sec to 5.4+/-0.06 sec and paw pinch threshold 
from 10.5+/-0.5 to 13.1+/-0.7. These differences were statistically 
significant (P&lt;05). 
The ability to induce analgesia with nicotine in transplanted animals was 
well maintained for at least up to 4 months. The differences between the 
pre- and post-nicotine pain sensitivities were statistically significant 
at all the tested time points for all three tests (P&lt;0.01). However, there 
appeared to be a slight decrement in response toward the end of the study, 
although this was not statistically significant. 
The sensitivity of the implanted chromaffin cells to low doses of nicotine 
was determined by using several doses of nicotine. Results are illustrated 
in FIG. 4. The lowest dose of nicotine, 0.05 mg/kg, produced a small, but 
statistically significant elevation in tail flick latency in animals with 
spinal cord bovine chromaffin cell implants (P&lt;0.05). This dose also 
appeared to produce an increase in paw pinch threshold and hot plate 
latency, but these were not statistically significant. At the highest dose 
of nicotine (0.2 mg/kg), the elevations in all three tests were nearly 
maximal (91% maximum tail flick latency, and 92% maximum paw pinch 
threshold). However, at this dose, there was also a small but significant 
elevation in the pain threshold of control animals. 
In order to determine the contribution of catecholamines and opioid 
peptides to the analgesia induced by nicotine in implanted animals, a 
group of animals with spinal cord bovine chromaffin cell implants was 
pretreated with either opiate antagonist naloxone, adrenergic antagonist 
phentolamine, or saline vehicle. These pre-injections did not alter pain 
sensitivity as determined 5 minutes after the injection (not shown). The 
injection of nicotine (0.1 mg/kg) in saline pretreated animals resulted in 
the usual induction of analgesia (FIG. 5). In contrast, this analgesia was 
severely attenuated in animals pretreated with naloxone as assessed by all 
three analgesiometric tests (P&lt;0.01). Phentolamine pretreatment completely 
blocked the elevation in hot plate latency (P&lt;0.01), and appeared to 
partially attenuate the elevation in tail flick latency and paw pinch 
threshold, but these were not statistically significant. 
EXAMPLE 2 
Adrenal tissue for transplantion was obtained from female Sprague-Dawley 
derived rats of the same group as the host animals. Adrenal medullary 
tissue was dissected from cortical tissue, cut into small pieces (less 
than 0.5 cu. mm.), and incubated in 2.5 S nerve growth factor (0.1 g/ml) 
in Hank's buffer containing 1 mg/kg bovine serum albumin) for 20 minutes. 
Tissue from one adrenal medulla was transplanted in each animal. Control 
animals received an equal volume of either heat killed adrenal medullary 
tissue or sciatic nerve tissue. Under pentobarbital anesthesia (30 mg/kg, 
i.p.), a laminectomy was performed to expose a 2-3 mm segment of the 
lumbar enlargement. Under a dissecting microscope, a small incision was 
made in the dura and pieces of adrenal medulla 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 returned to their cages 
for observation. Animals exhibiting motor abnormalities following surgical 
procedures were discarded from the study. 
Pain sensitivity was determined at several intervals following 
transplantation, but the 8 week time point is used here since 
morphological studies have shown that the grafts are well established by 
this time. Following determination of pain sensitivity in transplanted 
animals, response to nicotine stimulation (0.1 mg/kg, s.c.) was measured 
at 2, 10, 20, and 30 minutes following the injection. Results of this 
study are shown in FIG. 2. Animals receiving control transplants of either 
killed adrenal tissue or sciatic nerve tissue were pooled since there was 
no difference in their responses. Pain sensitivity in pre-implanted 
animals (not shown) was 3.2.+-.0.2 sec., 7.7.+-.0.8 sec., and 9.5.+-.0.5, 
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 implantation. Similarly, the injection of nicotine did not alter 
pain responsiveness in control transplant animals (FIG. 2). In contrast, 
this dose of nicotine produced potent analgesia in animals with spinal 
cord adrenal medullary transplants. The reduction in pain responsiveness 
was observed for all three analgesiometric tests (FIG. 2). This analgesia 
was apparent 2 minutes following nicotine stimulation, and pain threshold 
remained elevated for 10-20 minutes following the injection, tending 
toward baseline by 30 minutes. FIG. 6 shows the effect of antagonists 
(naloxone and phentolamine) on adrenal medulla transplanted animals. The 
results are similar to those observed with chromaffin cell implanted 
animals. 
In order to determine the underlying morphological changes responsible for 
the observed alterations in pain sensitivity, animals were prepared for 
electron microscopy following termination of behavioral testing. Animals 
were deeply anesthetized and perfused via the aorta with saline followed 
by buffered mixed aldehydes. The spinal cords were removed and processed 
for electron microscopy following standard procedure. 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. 7A and 7B). Ultrastructural 
observations revealed that the chromaffin cells in the transplants 
contained many chromaffin granules, primarily of the nonrepinephine 
containing type (FIG. 7B and 7D). The capillaries in the transplants 
appear to be fenestrated (FIG. 7C), in contrast to those of the host 
central nervous system, providing a potential for leakiness in the 
blood-brain barrier. It is noted that there did not appear to be extensive 
interaction between the graft tissue and the host centrall nervous system, 
although finger-like projections containing chromaffin granules can 
occasionally be seen protruding from chromaffin cells in the graft (FIG. 
7E). Thus it does not appear that synaptic relationships between the host 
and graft tissue are a necessary prerequisite for behavioral alterations. 
Rather, it is likely that the observed alterations in pain sensitivity are 
due to the humoral release of pharmacologically active substances into the 
subarachnoid space or cerebrospinal fluid in the CNS. 
The data presented herein clearly demonstrates that pain sensitivity, 
particularly to intractable pain as evidenced by hot-plate test, is 
alterable by implanting adrenal medullary tissue or isolated chromaffin 
cells into the subarachnoid space of the spinal cord. The ability of low 
dose of nicotine to induce analgesia in implanted animals suggests that 
the implanted material survives and retains functional ability to respond 
to nicotinic stimulation by releasing pain altering substances. Since this 
transplant is cross-species (bovine cells into rat CNS), it again 
emphasizes the immunological privilege of the CNS. Classical studies have 
suggested that the CNS is immunologically protected, and tissues with 
major histocompatibility differences transplanted to the CNS are rarely 
completely rejected. In support of this, Perlow et al., supra, have shown 
that dispersed, cultured bovine chromaffin cells survive at least 2 months 
without evidence of immunological rejection when transplanted to the 
cerebral ventricles of rats. In addition, these transplanted cells 
maintain the ability of synthesize and store catecholamines, as indicated 
by fluorescence histochemistry (not shown). 
These results further indicate that analgesia can be induced as early as 
one day following transplantation. The changes brought about by the 
implants appear to be maintained for at least 4 months since nicotine 
could still induce analgesia at this time. This suggests that neural 
tissue transplanted across species may provide a long-term therapeutic 
approach to treating intractable pain. 
The response to nicotine stimulation is dose-related, suggesting that the 
implants respond to higher doses with an increased release of nicoceptor 
interacting substances. At the highest dose of nicotine, the increase in 
pain threshold was nearly maximal for the tests employed. However, the 
intermediate dose of 0.1 mg/kg was determined to be optimal for these 
studies, since this dose by itself does not alter pain sensitivity in 
these animals. 
The ability of naloxone to block the analgesia normally induced by nicotine 
in the implanted animals supports the notion that this analgesia is the 
result of the release of opioid peptides from the transplanted chromaffin 
cells. The partial attenuation by phentolamine suggests that catecholamine 
release may also be involved. It is possible that other neuropeptides in 
the chromaffin cells may also be involved. Thus, the co-release of two or 
more pharmacologically active agents, such as norepinephrine and 
enkephalin, from implanted chromaffin cells would act synergistically to 
produce paian relief. Whatever the mechanism, the results clearly 
demonstrate the efficacy of the methods described herein for relieving 
and/or modulating sensitivity to pain. The technique disclosed herein 
provides a new therapeutic approach for the relief of intractable pain. 
It is understood that the examples and embodiments described herein are for 
illustrative purposes only and that various modifications or changes in 
light thereof will be suggested to persons skilled in the art and are to 
be included within the spirit and purview of this application and the 
scope of the appended claims.