(The FASEB Journal. 1999;13:S277-S280.)
© 1999 FASEB
Fine structure of host-graft relationships between transplanted chromaffin cells and CNS
GEORGE D. PAPPAS1
The Psychiatric Institute, Department of Psychiatry and Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, Illinois, USA
1Correspondence: The Psychiatric Institute and Department of Cell Biology, University of Illinois at Chicago, 1601 West Taylor St (M/C 912), Chicago, IL 60612 USA. E-mail: gdpappas{at}uic.edu
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ABSTRACT
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Our laboratory studies have shown that transplantation of adrenal
medullary tissue or isolated chromaffin cells into central nervous
system (CNS) pain modulatory regions (i.e., periaqueductal gray and
subarachnoid lumbar spinal cord) can reduce pain sensitivity of rats in
both acute and chronic pain. The analgesia produced by these
transplants is thought to result from release of both opiate peptides
and catecholamines. Morphologically, these animal studies also suggest
that there is no development of tolerance over long periods of time,
and the transplanted chromaffin cells appear to be robust and well
integrated with the host tissue. In our initial clinical studies, where
allografts of adrenal medullary tissue were transplanted intrathecally
to relieve intractable cancer pain, patients obtained significant and
long-lasting pain relief. Increased cerebrospinal fluid (CSF) levels of
metenkephalin were correlated with the decreased pain scores. Histology
of autopsy tissue obtained from two patients with 1 year transplants
revealed viable transplanted chromaffin cells. Because of the limited
availability of human adrenal glands, sources of xenogeneic chromaffin
cells will need to be identified if effective transplantation therapy
for chronic pain is to be developed further.—Pappas, G. D. Fine
structure of host-graft relationships between transplanted chromaffin
cells and CNS.
Key Words:
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INTRODUCTION
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ALTHOUGH NEURONAL TRANSPLANTATION into the central
nervous system (CNS) has been used to replace or repair degenerative
brain tissue (e.g., in Parkinson’s disease), we have used chromaffin
cells to augment the normal production of neuroactive substances. Our
laboratory at the University of Illinois at Chicago has shown in both
acute and chronic pain models that transplantation of chromaffin cells
into the neuraxis in the pain modulatory regions can drastically reduce
pain sensitivity in rodents (1)
. Chromaffin cells of the
adrenal medulla secrete a large number of neuroactive substances,
including catecholamines, enkephalins, and many other neuropeptides
(2
, 3)
. When transplanted into the CNS, they function as a
dynamic biological ‘reservoir’, providing an endogenous source of
these continuously renewable neuroactive molecules. The analgesia
produce by these transplants probably result from the release of both
opioid peptides and catecholamines, because opiate and adrenergic
antagonists attenuate or even block the analgesic effect. There is no
decrement over time (1 year in rats) in the magnitude of chronic pain
relief in these animal studies, suggesting that transplanted chromaffin
cells can reduce chronic pain without the development of appreciable
tolerances (4)
.
In our rat studies, we used adrenal allografts to transplant chromaffin
cells as dissected intact tissue pieces of adrenal medulla or isolated
chromaffin cells. Transplants were placed either intraparenchymally in
the periaqueductal gray or extraparenchymally into the subarachnoid
space on the surface of the lumbar spinal cord. No immunosuppression
was used (5)
. Electron microscopy revealed intact and
viable transplants, while behavioral testing demonstrated the analgesic
properties of the allografts.
Apart from the chromaffin cells, a plasma cell infiltrate can be seen
on histological study of the allografts after several months
(5)
. These plasma cells of the host appear to have
extensively organized ribosome-studded endoplasmic reticulum indicating
active protein synthesis (see Fig. 1
,
2
). The significance of the plasma cell infiltrate is
uncertain, although it is known that allograft tolerance may be
involved by anti-idiotypic antibodies secreted by host plasma cells
(6)
.
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Figure 1. Electron micrograph of a portion of a 12-wk-old adrenal medullary
allograft in the periaqueductal gray of a rat. Plasma cells (P) of host
origin can be seen in the implant area containing chromaffin cells
(CH). B, blood vessel.
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Figure 2. As in Fig. 1
but at higher magnification. An electron micrograph of a
portion of a grafted chromaffin cell (CH) in very close proximity with
a host plasma cell (PL). Dilated elements of the endoplasmic reticulum
can be seen in the cytoplasm of the plasma cell. Note that between
arrows, a very fine neural process can be clearly seen intervening
between these two cells. The close proximity of robust plasma cells of
the host with the implanted chromaffin cells suggest that these plasma
cells may be secreting idiotypic antibodies that promote and support
the long-term survival of these allografts.
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The transplantation of isolated bovine chromaffin cells into the rat
CNS (i.e., xenografts), on the other hand, required immunosuppression
(i.e., cyclosporin A, which blocked T-helper lymphocyte function)
(8)
. We found that daily immunosuppression for ~3 wk was
necessary for the same long-term survival that we obtained with
allografts (7)
. We postulated that short-term
immunosuppression is probably necessary after the surgical trauma and
the resultant transient disruption of the blood-brain barrier at the
injection sites. We have also shown that chromaffin cells do not
express the major histocompatibility complex antigens (MHC I and II)
(8)
. Therefore, the isolation of chromaffin cells from the
other cellular components in the adrenal medullary tissue (i.e.,
fibroblasts, smooth muscle, and highly immunogenic endothelial cells)
is probably necessary to achieve long-term survival of the xenogenic
chromaffin cells. Older isolation procedures of chromaffin cells from
other tissue components produced ~95% pure chromaffin cell yield
(9)
. The 5% nonchromaffin ‘passenger’ cells elicit a
vigorous host immune response, which may bring about the rejection of
both passenger and also chromaffin cells. Recently, we have developed a
more highly purified preparation with yields of >99.5% chromaffin
cells. Preliminary findings indicate that nonimmunosuppressed rats
become analgesic when these highly purified bovine cells are grafted
onto the lumbar spine (10)
.
In addition to the analgesimetric findings that suggest graft
functional survival, more direct morphological findings clearly show
that host neural processes are in close contact with the grafted
chromaffin cells. In fact, many neuronal processes form what
structurally appear to be synaptic contacts onto chromaffin cells
(9
, 11)
. These presynaptic-appearing processes have small
clear vesicles (Fig. 3
), which appear to be similar to the postganglionic
cholinergic synapses found in situ in the adrenal medulla.
Occasionally, larger dense-core vesicles may be present in these
presynaptic-appearing processes. We have also reported earlier that
host neuronal processes have the appearance of being postsynaptic and
can sometimes be found to be in contact with chromaffin cells
(1)
. By anterograde and retrograde tracing, the origin of
these neuronal processes, which make synaptic-appearing contacts on the
grafted chromaffin cells, appear to originate from interneurons of the
host parenchymal tissue. Some endings were found to arise in afferent
neuronal processes from more distant areas that normally project into
the transplant region (12)
. Presynaptic-appearing neuronal
processes are also found on chromaffin cells implanted
extraparenchymally in the subarachnoid space on the surface of the
lumbar spinal cord. We have no evidence that these neuronal processes
originate from the spinal cord itself; rather, it seems that these
fibers are from the nearby arachnoid granulation (13)
.
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Figure 3. Electron micrograph showing a portion of a bovine chromaffin cell (N,
nucleus of chromaffin cell) implanted on the surface of the lumbar
spinal cord in the subarachnoid space of a rat. Note that a neuronal
process of the host containing vesicles (V) has formed a synaptic-like
contact with the xenogeneic chromaffin cell.
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Nerve growth factor (NGF) induces chromaffin cells to change shape,
become elongated, and send out fine processes (14)
.
Placing an Alza pump containing NGF near where the chromaffin cell
transplant is located causes long cytoplasmic fiber-like extensions to
develop from the chromaffin cell extensions (see Fig. 4
). Although there is no direct evidence that these synapses are
functionally integrated with the host CNS, the presence of structurally
specialized areas of contact between host and graft (either allogeneic
or xenogeneic) suggests the possibility.
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Figure 4. Electron micrographs of bovine chromaffin cells implanted in the
periaqueductal gray of a rat. An Alza pump containing nerve growth
factor (NGF) was also placed nearby causing the chromaffin cells to
change shape from cuboidal to having long cytoplasmic processes (at
arrowheads in the above micrograph and at opposing double arrows in the
lower one). Also note in the lower micrograph that one of these
long-extended chromaffin cell processes (double arrows) shows two areas
where host processes containing vesicles (V) form what appears to be a
synaptic contact onto this xenogeneic cytoplasmic process.
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As a logical extension of our animal studies, we turn to the
clinical problems posed by the development of tolerance to opiates in
humans with intractable cancer pain. Most patients develop tolerance to
opioids, and as the dosage is increased, undesirable complications
develop. Analgesic effectiveness becomes minimal, if at all. Surgical
implantation of epi- or subdural cannular ports and pumps may partially
solve this problem. However, the dosage of infused drug is usually not
optimal, and, in addition, morbid complications commonly occur,
including mechanical malfunctions and infections (15)
.
With these concerns and the findings of our animal studies, the
potential benefit of transplanting chromaffin cells as an endogenous
source of pain-reducing substances as a long-term or permanent solution
to stabilize, reduce, or eliminate exogenous opioid administration
became apparent. The clinical studies were initiated with Dr. Alon P.
Winnie at Cook County Hospital and colleagues at the Pain Control
Center of the University of Illinois Hospital, Chicago
(16)
. We have also enlarged our studies with Dr. Yves
Lazorthes and colleagues at the University Paul Sabatier in Toulouse,
France (17)
. Donor human adrenal medullary tissue
allografts were implanted into the subarachnoid space on the surface of
the lumbar spinal cord in 20 patients. Our initial findings indicated
that this method might be a valuable approach for the alleviation of
chronic pain (18)
. In general, patients showed a
significant reduction in their pain scores that was directly correlated
with a large increase in metenkephalin in the CSF (13
, 17
, 18)
. Less than half of the patients no longer required narcotic
intake. More significant, however, was the rest of the patients who
completed the study completely stabilized their narcotic intake at
minimal does (18)
. We concluded that no opiate tolerance
develops in the presence of chromaffin cell transplants. Lastly,
autopsy material from two patients with 1 year transplants demonstrated
chromaffin cells in situ; grafted tissue fragments were
positively stained with specific chromaffin cell markers (i.e.,
tyrosine hydroxylase, dopamine-ß-hydroxylase, and chromogranin A),
indicating viability of the graft (19)
.
In summary, our animal and preliminary clinical studies indicate that
chromaffin cell transplants might be effective in controlling chronic
pain. Future clinical work will include controlled comparisons with
placebos. In addition, given the limited availability of human adrenal
glands (allografts), we plan to also assess the clinical usefulness of
xenogeneic chromaffin cells. Because chromaffin cells do not by
themselves appear to activate the host immune system, refinements in
purification of the cells will allow for effective transplantation
therapy for intractable pain in the absence of morbid immunosuppressive
drugs.
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ACKNOWLEDGMENTS
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I would like to thank Professor Raymond Pollack, Chief,
Transplantation Surgery, College of Medicine, University of Illinois at
Chicago, for his advice and critical reading of this paper. This work
was supported by NIH Grant NS-28931.
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REFERENCES
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ABSTRACT
INTRODUCTION
