John D. Ortega

Short-term immunosuppression enhances long-term survival of bovine chromaffin cell xenografts in rat CNS.

Xenogeneic donors, a largely untapped resource, would solve many of the problems associated with the limited availability of human donor tissue for neural transplantation. Previous work in our laboratory has revealed that xenografts of isolated bovine chromaffin cells survive transplantation into the periaqueductal gray (PAG) of immunosuppressed adult rats. Electron microscopic analysis reveals that graft sites contain healthy chromaffin cells, but do not contain host immune cells typical of graft rejection. The aim of the current study was to assess the necessary conditions for long-term survival of bovine chromaffin cell xenografts in the central nervous system (CNS). In particular, the need for short-course vs. permanent immunosuppressive therapy with cyclosporine A (CsA) for the long-term survival of grafted bovine chromaffin cells was addressed. Grafts from animals receiving continuous CsA treatment for either 3, 6, or 12 wk contained large clumps of dopamines-β-hydroxylase (DBH) positive cells in contrast to the few surviving cells observed in nonimmunosuppressed animals. In addition, grafts from animals that had CsA treatment terminated at 3 or 6 wk contained similarly large clumps of DBH-positive cells. Furthermore, short-term immunosuppression (3 wk) appeared to enhance the long-term survival of grafted cells, since clumps of DBH staining cells could still be positively identified in the host PAG at least 1 yr after transplantation. Complete rejection of graft tissue depends on several factors, such as blood–brain barrier integrity, the presence of major histocompatability complex (MHC) antigens in either the host or graft, and the status of the host immune system. By using a suspension of isolated bovine chromaffin cells, potential MHC antigen presenting cells, such as endothelial cells, are eliminated. In addition, CsA treatment may negate the immunologic consequences of increased blood–brain barrier permeability following surgical trauma by attenuating the host cell mediated response. In summary, long-term survival of isolated chromaffin cell xenografts in the rat CNS may be attained by a short-term course of CsA.

Host-graft relationships of isolated bovine chromaffin cells in rat periaqueductal grey

SummaryThe transplantation of chromaffin cells from the adrenal medulla into pain modulatory regions of the CNS has previously been shown to reduce pain sensitivity, most likely via local release of neuroactive substances from the transplanted cells. The ready availability of bovine adrenal glands, as well as the high levels of opioid peptides produced by their chromaffin cells, make these glands a potentially valuable donor source for antinociception studies. However, the success of these xenografts depends on their ability to survive and integrate within the host CNS.The aim of the present study was to assess host-graft relationships of bovine chromaffin cells transplanted to the rat CNS. We have found that isolated bovine chromaffin cells survive for at least three months in the rat periaqueductal grey, with no evidence of immunological response following a short-term course of immunosuppressant treatment. In the early stages following transplantation, only minor pathology is found at the injection site, which apparently recovers completely at later stages. The host-graft borders are not well demarcated, in contrast to solid tissue grafts.Neuronal processes of host origin, forming numerous synapses with the transplanted bovine chromaffin cells, are apparent by three weeks following transplantation. Migration also occurs from the graft into the host parenchyma, as evidenced by individual chromaffin cells found near host parenchymal blood vessels. The clusters of chromaffin cells found in the graft itself are generally not very vascular, in contrast to solid tissue grafts. The chromaffin cell clusters are surrounded by blood vessels of the non-fenestrated CNS type at the host-graft border. It is likely that the small size of the graft does not require extensive angiogenesis. The lack of fenestrated peripheral-type endothelial capillaries, normally seen in adrenal medullary tissue grafts, may contribute to the survival of these xenografts in the rat brain.

Pharmacologic characterization of opioid peptide release from chromaffin cell transplants using a brain slice superfusion method

A simple chamber and an inexpensive superfusion system for studying mammalian brain slices containing neural transplants is described. With this method, rat brain slices containing bovine chromaffin cell transplants can be maintained for several hours, allowing for the determination of neurochemical characteristics and pharmacologic responsiveness of the grafted cells. Using this technique, basal and nicotine-stimulated release of metenkephalin from rat periaqueductal gray slices containing bovine chromaffin cell transplants were measured. Results showed that met-enkephalin release can be increased by nicotinic stimulation in slices containing chromaffin cell, but not control implants, for at least 8 weeks postimplantation. Furthermore, this response was doserelated. These results are in good agreement with previous behavioral studies and provide corroborative evidence for the mechanism of pain reduction by the release of opioid peptides from chromaffin cell transplants in the periaqueductal gray. This study demonstrates that neurochemical and pharmacologic analyses of neural transplants using a superfused brain slice method can be a complementary approach in determining the underlying mechanisms of neural transplants in the central nervous system.

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

Abstract: 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.

Neuropeptide and Catecholamine Delivery to Central Nervous System by Implanted Chromaffin Cells

Publisher Summary This chapter explores neuropeptide and catecholamine delivery to central nervous system (CNS) by implanted chromaffin cells. For the purpose of providing local CNS access to catecholamines and neuropeptides for therapeutic application, both allogeneic (between individuals of the same species) and xenogeneic (between donor and host of different species) sources have been used. Allogeneic sources are particularly important as this mimicks the most likely scenario for initial clinical application. Preliminary clinical trials for alleviation of chronic pain in terminal cancer patients utilized adrenal medullary tissue from human organ donors. However, although human adrenal medullary allografts are the most likely sources for initial clinical studies, their limited availability and nonuniformity reduce their feasibility for widespread use. Xenogeneic donors are a potential alternative, and largely untapped, source of chromaffin cells for transplantation. This chapter describes the preparation and use of both adrenal medullary allografts and xenografts as an implantable local delivery source of catecholamines and opioid peptides to the CNS. Results from the methods presented in this chapter indicate that chromaffin cells can serve as a source of biologically active substances such as opioid peptides and catecholamines when transplanted into appropriate CNS sites.

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.

Non-Chromaffin Cell Constituents of the Adrenal Medulla are Detrimental to the Survival of Grafted Adrenal Chromaffin Cells: Studies in Rats and Non-Human Primates

The initial rationale for using adrenal chromaffin cells in transplantation experiments was to provide a paraneural source of dopamine to replenish the dopamine insufficiency created in animal models of parkinsonism and idiopathic Parkinson’s disease. Intraventricular transplants of adrenal medulla (AM) survive, synthesize and secrete eatecholamines, and reverse drug-induced motor asymmetries in unilateral nigrostriatal lesioned rats. Subsequent studies in which AM grafts were placed directly into the striatum also induced functional recovery, albeit partially, and short-lived. It soon became increasingly clear that ehromaffin cells survived poorly within the striatal parenchyma. Studies carried out in non-human primates and autopsy eases from AM-grafted parkinsonian patients confirmed the notion that adrenal ehromaffin cells do not survive well following intrastriatal transplantation. The use of trophic factors to improve chromaff cell survival heralded the second era of AM transplantation. Injections of /3. nerve growth factor into AM graft sites augments chromaffin cell survival, induces morphological differentiation, and increases the magnitude and duration of functional effects. Chromaffin cell survival can also be increased by cografting AM with growth factor-producing C6 gliomas, astrocytes genetically engineered to produce/3NGF, and transected peripheral nerve whose damaged Schwann cells secrete a variety of trophic molecules including/3NGF. We have begun to utilize an alternative approach towards enhancing chromaffin cell survival. Instead of using trophic molecules to enhance graft viability we have attempted to minimize factors which may negatively impact upon chromaffin cell survival and induce AM grafts to degenerate. Adrenal chromaffin cells have previously been demonstrated to survive well following implantation into the periaqueductal gray once the chromaffin cells were isolated from nonchromaffin cells found in the AM. These data suggest that fibroblasts, blood-borne leukocytes, and/or endothelial cells within the AM may be detrimental to chromaffin cell viability and, at least in part, underlie the poor survival of these cells following intrastriatal implantation. We presently assessed the effects of isolating bovine ehromaffin cells from the other cell types within the AM upon ehromaffin cell graft survival in rats and MPTP treated rhesus monkeys following intrastriatal transplantation. Three groups of immunosuppressed rats were employed. Group 1 received grafts of bovine AM following gland perfusion and dissection of the medulla from the adrenal cortex. Group 2 received implants of isolated adrenal chromaffin cells. Chromaffin cells were isolated in vitro by placing them in a Pereoll gradient resulting in the segregation of dead cells, residual cortical cells, red blood cells, and viable medullary cells. Once separated, the medullary cells were differentially plated resulting in a 95% pure population of chromaffin cells. Group 3 received implants of isolated adrenal chromaffin cells which were reseeded with fibroblasts and endothelial cells which were allowed to proliferate in culture. All rats were sacrificed 1-2 months following transplantation. Rats in Group 1 displayed viable tyrosine hydroxylase (TH) and dopamine /3 hydroxylase (D/3H)-immunoreactive (ir) transplants. However, these grafts tended to be small, the graft-host interface was infiltrated with macrophages, and many of the chromaffin cells appeared to be in the process of degenera-