Transplanted Chromaffin Cells Research Articles

Re-establishment of neurochemical coding of preganglionic neurons innervating transplanted targets

We investigated the effect on neurochemical phenotype of changing the targets innervated by sympathetic preganglionic neurons. In neonatal rats, the adrenal gland was transplanted into the neck, to replace the postganglionic neurons of the superior cervical ganglion. Transplanted adrenal glands survived, and contained noradrenergic and adrenergic chromaffin cells, and adrenal ganglion cells. Retrograde tracing from the transplants showed that they were innervated by preganglionic neurons that would normally have supplied postganglionic neurons of the superior cervical ganglion. The neurochemical phenotypes of preganglionic axons innervating transplanted chromaffin cells were compared with those innervating the normal adrenal medulla or superior cervical ganglion neurons. As in the normal adrenal gland, preganglionic nerve fibres apposing transplanted chromaffin cells were cholinergic. The peptide and calcium-binding protein content of preganglionic fibres was similar in normal and transplanted adrenal glands. In both cases, cholinergic fibres immunoreactive for enkephalin targeted adrenergic chromaffin cells, whilst cholinergic fibres with co-localised calretinin-immunoreactivity innervated noradrenergic chromaffin cells and adrenal ganglion cells. In contrast to the innervation of normal adrenal glands, these axons lacked immunoreactivity to nitric oxide synthase. In a set of control experiments, the superior cervical ganglion was subjected to preganglionic denervation in rat pups the same age as those that received adrenal transplants, and the ganglion was allowed to be re-innervated over the same time course as the adrenal transplants were studied. When the superior cervical ganglion was re-innervated by preganglionic nerve fibres, we observed that all aspects of chemical coding were restored, including cholinergic markers, nitric oxide synthase, enkephalin, calcitonin gene-related peptide and calcium binding proteins in predicted combinations, although the density of nerve fibres was always lower in re-innervated ganglia. These data show that the neurochemical phenotypes expressed by preganglionic neurons re-innervating adrenal chromaffin cells are selective and similar to those seen in the normal adrenal gland. Two explanations are advanced: either that contact of preganglionic axons with novel target cells has induced a switch in their neurochemical phenotypes, or that there has been target-selective reinnervation by pre-existing fibres of appropriate phenotype. Regardless of which of these alternatives is correct, the restoration of normal preganglionic codes to the superior cervical ganglion following denervation supports the idea that the target tissue influences the neurochemistry of innervating preganglionic neurons.

Fine structure of host‐graft relationships between transplanted chromaffin cells and CNS

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.

Attenuation of NMDA-induced spinal hypersensitivity by adrenal medullary transplants

Abnormal sensory hyperexcitability consequent to peripheral injury most likely involves activation of N-methyl- d-aspartate (NMDA) receptors in the spinal cord. This activation may lead to a cascade of neuroplastic events resulting in the exaggeration of sensory responses and the persistence of pathological pain states. Recent studies in our laboratory have demonstrated that the transplantation of adrenal medullary cells into the spinal subarachnoid space can alleviate pathological pain symptoms, possibly by reducing spinal hyperexcitability. The purpose of this study was to assess spinal NMDA activation-induced hypersensitivity to noxious and innocuous stimuli, and determine whether adrenal medullary transplants can intervene favorably to reduce these responses. Animals with either adrenal medullary or control transplants were injected intrathecally with several doses of NMDA, and responses to sensory stimuli were determined over time. NMDA at all doses tested (1–50 nmol) produced significant thermal and mechanical hyperalgesia and tactile allodynia in control transplanted animals, with peak severity at 30 min post-injection. In contrast, both the severity and duration of these exaggerated sensory responses were markedly reduced in animals with adrenal medullary transplants. To assess a possible contribution of released opioid peptides and catecholamines from the transplanted chromaffin cells, animals were pretreated with opiate antagonist naloxone or α-adrenergic antagonist phentolamine. While naloxone was ineffective, the phentolamine partially attenuated, but did not completely abolish, the antinociceptive effects of the transplants. The results of these studies demonstrate that adrenal medullary grafts can reduce hypersensitivity responses to NMDA-mediated activation via α-adrenergic modulation in addition to other neuroprotective mechanisms.

Influence of the CNS Environment on Chromaffin Cell Survival and Catecholamine Secretion Patterns

Chromaffin cells implanted into the CNS have been used as a potential source of sustained catecholamine delivery, although their survival and continued catecholamine secretion are controversial. In addition, chromaffin cells exhibit a high degree of neurochemical plasticity in response to environmental factors. The present aims were to determine whether the CNS provides a supportive environment for sustained catecholamine production in transplanted chromaffin cells and to assess whether this novel environment alters patterns of catecholamine secretion. Catecholamine release from bovine chromaffin cells implanted into the rat midbrain was determined in brain slices. In addition, alterations in catecholamine secretion patterns, particularly adrenaline/noradrenaline ratios, were compared in vitro versus in transplants. Results indicated that brain slices containing chromaffin cell implants released high basal and nicotine-stimulated levels of adrenaline and noradrenaline. It is surprising that although adrenaline/noradrenaline ratios steadily declined in culture, this did not occur when cells were transplanted to the CNS in the early postharvesting phases. However, if cells were transplanted following longer periods in culture, adrenaline/noradrenaline ratios remained low. Together, these results suggest that the CNS can provide a supportive environment for chromaffin cell survival and that the pattern of catecholamine secretion can be optimized by prior in vitro manipulation.

Optimization of adrenal medullary allograft conditions for pain alleviation.

Previous findings in our laboratory have demonstrated that transplants of adrenal medullary tissue into the spinal subarachnoid space can alleviate pain, most likely via sustained local release of pain-reducing neuroactive substances from the transplanted chromaffin cells. The success of this work in animal models has led to preliminary clinical trials with promising results. However, before large-scale clinical studies are undertaken, numerous issues should be resolved, many of which can be readily addressed initially in the laboratory. One of these is the amount of donor adrenal medullary tissue necessary to produce long-term antinociception. Although tissue from two adrenal glands has generally been used, it is unknown whether less would be equally effective, or more could increase analgesic potency. To address this, various amounts of adrenal medullary tissue, ranging from one to ten donor glands, were used. Results showed lowered antinociceptive benefits when only one adrenal medulla was used, but only small and short-lived increases when donor material was increased substantially. In addition, assays of atecholamine and Met-enkephalin release in the host spinal CSF revealed only slight further increases following the transplantation of more than 2-4 glands. These results indicate that a small amount of adrenal medullary tissue is necessary and sufficient to produce sustained antinociception, and suggest that higher amounts may result in tolerance development or feedback inhibition. Another important issue is the ability to retain antinociceptive potency if donor tissue is maintained in culture prior to transplantation, since the coordination of donor harvesting and recipient availability is often difficult. To address this, donor adrenal medullary tissue was maintained in explant culture for various periods following dissection. Results indicated that adrenal medullary tissue can be maintained in culture for up to 30 days prior to transplantation without decrement in antinociceptive potency, and that a period of 7-21 days results in improved graft viability. Results of this study indicate that some of the critical issues for successful neural transplantation outcomes can be initially addressed in pre-clinical studies.

Origin of afferent projections into bovine chromaffin cell implants in the rat periaqueductal gray determined by retrograde and anterograde tracing.

We have previously described long-term survival of isolated bovine chromaffin cell suspension grafts in the periaqueductal gray of adult rats. Electron microscopic analysis of the graft sites revealed synapses on the transplanted chromaffin cells. The origin of these synapses is not known, but they are probably derived from the host since the initial grafts were suspensions of chromaffin cells that were essentially free of other cell types.In order to determine the origin of the observed synapses, retrograde and anterograde tracer analyses, were performed on grafted rats at 4 and 8 weeks after transplantation. Following injection of the retrograde tracer (Fluoro-Gold) into graft sites, four major host sites were labeled: hindbrain reticular formation, substantia nigra, lateral hypothalamus, and cingulate cortex. Injection of anterograde tracer (rhodamine-conjugated dextranamine) into the substantia nigra, lateral hypothalamus, and cingulate cortex produced labeled fibers and terminals in and around 4 and 8 week old chromaffin cell graft sites. An increase in both the number of retrogradely labeled cells, as well as in the density of anterogradely labeled fibers and terminals within the graft site, was observed from 4 to 8 weeks.This study shows that graft innervation from the host is primarily from areas that normally project afferent fibers to the periaqueductal gray. The increase in labeled fibers, and terminals over 8 weeks suggests that de novo synapse formation on grafted bovine chromaffin cells is a continuous process that is dependent on the regenerative capacity and plasticity of the host neuronal network and the grafted bovine chromaffin cells.

Mouse adrenal chromaffin cells can transform to neuron-like cholinergic phenotypes after being grafted into the brain.

The development of neuron-like cholinergic immunophenotypes by adrenal chromaffin cells was studied in 10-week-old mouse adrenal medullary grafts. Fragments of chromaffin tissue were implanted into mouse hippocampus, and antibodies specific for neurofilaments (NF), neuron-specific enolase (NSE), choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and phenylethanolamine-N-methyltransferase (PNMT) were applied to the grafts. Adrenal medulla grafts survived well and most of the transplanted cells were either round or polygonal. A minority of chromaffin cells elaborated an intermediate or sympathetic neuron phenotype. Chromaffin cells showed pronounced immunoreactivity for NSE in their perikarya and axon-like processes: immunoreactivity for NF was only found in a few processes. In adjacent immunohistochemically stained sections, the transplanted cells stained for ChAT and AChE. At the electron-microscope level, the immunohistochemical reactions for the two acetylcholine-related enzymes were mainly located on the endoplasmic reticulum and in cell processes. Immunoreactivity for PNMT was found to decline in transplanted chromaffin cells below that of normal adrenal medulla. These observations suggest that, in adrenal medullary grafts implanted into the hippocampus, chromaffin cells are endowed with neuron-like cholinergic immunophenotypes.

Transplants of immunologically isolated xenogeneic chromaffin cells provide a long-term source of pain-reducing neuroactive substances

Adrenal medullary chromaffin cells are a potential source of neuroactive substances for transplantation into the CNS to alleviate neurochemical deficits. In particular, work in our laboratory has suggested that adrenal medullary transplants in the spinal subarachnoid space can alleviate pain by providing sustained local delivery of catecholamines and opioid peptides. One of the major limitations for clinical application of neural transplantation is the availability of donor material in sufficient quantities. This limitation may be overcome by the use of xenogeneic donors if long-term graft rejection can be prevented. The purpose of this study was to assess whether xenogeneic chromaffin cells immunologically isolated by semipermeable membranes could survive and continue to reduce pain when transplanted into the CNS. Isolated bovine chromaffin cells were encapsulated by semipermeable polymer membranes and implanted into the rat spinal subarachnoid space. Pain sensitivity was assessed at several intervals up to 3 months following implantation. Results indicated that encapsulated bovine chromaffin cell implants, but not empty control capsules, could repeatedly reduce pain sensitivity with nicotine stimulation for the duration of the study. This response was dose related, indicating that pharmacologic integrity of the transplanted chromaffin cells is retained. The analgesia induced by encapsulated chromaffin cell implants could be attenuated by the opiate antagonist naloxone and the alpha-adrenergic antagonist phentolamine, suggesting the involvement of both opioid peptides and catecholamines in mediating this response. In addition, in vitro neurochemical studies of recultured capsules revealed sustained release of Met-enkephalin and catecholamines from encapsulated cells 3 months following implantation into the spinal subarachnoid space.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell adhesion molecules in adrenal medulla grafts: Enhancement of chromaffin cell [formula omitted] expression and reorganization of extracellular matrix following transplantation

Intracerebral adrenal medulla grafts have been used in human patients as an experimental treatment for Parkinson's disease, based on studies in animal models of this disorder. However, alterations in chromaffin cell properties after transplantation and the factors controlling graft survival are poorly understood. Since cell adhesion molecules (CAMs) are involved in regeneration and development of neural tissue in vivo and in vitro, the present study was undertaken to determine the expression of CAMs in adrenal medulla isografts. Fragments of rat adrenal medulla were implanted into the right lateral ventricle. The majority of grafts survived quite well, for up to 2 months (the longest studied period). The implanted chromaffin cells did not develop extensive processes. The cells retained tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) immunoreactivity, while phenylethanolamine N-methyltransferase (PNMT) expression was decreased. Surviving transplanted chromaffin cells showed enhancement and spreading of surface L1 Ng-CAM expression as compared to normal chromaffin cells in adrenal medulla. The implanted chromaffin cells demonstrated only partial conversion to neuronal phenotypes. These chromaffin cells did not develop extensive processes, but showed an enhancement of L1 Ng-CAM expression. Surviving chromaffin cells were accompanied by reorganization of their closely associated extracellular matrix (ECM). As compared to normal in situ adrenal medulla, graft ECM demonstrated a substantial increase of L1 Ng-CAM and laminin immunoreactivities and a distinct decrease in J1/tenascin expression. Some adrenal medulla grafts degenerated, particularly when misplaced within the host brain parenchyma. In these cases the grafts showed fragmentation of ECM and gradual disappearance of CAMs. These results suggest that surviving adrenal medulla grafts exhibit increased synthesis of certain CAMs by chromaffin cells, which may be involved in interactions between chromaffin cells and the surrounding ECM. It is speculated that both surviving and degenerating adrenal medulla grafts could provide CAMs and ECM components including laminin to host brain and this way contribute to functional effects of grafts.

Adrenal medullary implants in the rat spinal cord reduce nociception in a chronic pain model

Previous work in this laboratory has indicated that the transplantation of adrenal medullary tissue into the subarachnoid space of the rat spinal cord can reduce pain sensitivity to acute noxious stimuli, particularly following stimulation by nicotine. This most likely results from the stimulated release of opioid peptides and catecholamines from the transplanted chromaffin cells. However, chronic pain models may more closely resemble human clinical pain, and the arthritic rat model has been used for screening potential therapeutic strategies. The purpose of the present study was to assess the potential for adrenal medullary tissue implanted into the spinal subarachnoid space to alleviate chronic pain. Adrenal medullary tissue was implanted into adjuvant-in-duced arthritic rats, and changes in body weight and vocalization responses were monitored over the 10 week course of the disease. Results indicate that the severe weight reduction normally associated with this inflammatory arthritis was attenuated by adrenal medullary, but not control, implants. In addition, vocalizations were reduced in animals implanted with adrenal medullary, but not control tissue following nicotine stimulation. This reduction was blocked by the opiate antagonist, naloxone, and partially attenuated by the alpha-adrenergic antagonist, phentolamine. Together, these results suggest that the transplantation of adrenal medullary tissue into the subarachnoid space of the spinal cord may provide a local source of opioid peptides and catecholamines for the reduction of chronic pain.

Adrenal chromaffin cells as transplants in animal models of Parkinson's disease.

The field of neural transplantation has moved rapidly forward in the last decade. Initially, fetal cells were used as implants to investigate their potential to ameliorate deficits in animal models of Parkinson's disease. However, because of the moral and legal problems associated with the use of fetal tissues in humans, alternative sources of donor tissue were sought which possessed the structural and functional characteristics needed to improve motor function in Parkinsonian patients. To date, one of the most promising tissues being investigated is the adrenal medulla, whose chromaffin cells possess an inherent plasticity of form and function. Transplanted chromaffin cells currently are being studied by a variety of approaches, including electron microscopy, in mouse, rat, and primate models of Parkinson's disease. An overview of the role of the chromaffin cell in this exciting and clinically important arena is briefly reviewed, with an emphasis on the fine structure of implanted chromaffin cells.