Prolonged survival of bovine adrenal chromaffin cells in rat cerebral ...

Proc. Nati. Acad. Sci. USA Vol. 77, No. 9, pp. 5278-5281, September 1980

Cell Biology

Prolonged survival of bovine adrenal chromaffin cells in rat cerebral ventricles (adrenal medulla/transplantation/catecholamine/neuroendocrine)

M. J. PERLOW*, K. KUMAKURAt, AND A. GUIDOTTI Laboratory of Clinical Psychopharmacology and Laboratory of Preclinical Pharmacology, National Institute of Mental Health,

St. Elizabeth's Hospital, Washington, D.C. 20032

Communicated by Marshall Warren Nirenberg, May 27, 1980

a pellet was obtained (3 X 106 cells per ml). Small volumes (0.025 ml in the adult and 0.01 ml in the neonate) of this pellet were injected into the right cerebral ventricle of neonatal (0-18 hr old; freehand injection with a 21-gauge needle through the scalp) and adult [500-600 g; stereotaxically as described (4)] Sprague-Dawley rats. After the operation, the animals were given 30,000 units of penicillin subeutaneously. The rats were sacrificed approximately 10, 30, and 60 days after transplantation. Their brains were rapidly removed and frozen on dry ice. Coronal sections (15 ,tm) from the brain were obtained and stained for catecholamine histofluorescence with glyoxylic acid (9). The same sections were later stained with cresyl violet.

ABSTRACr

Dispersed, cultured bovine adrenal chromaffin cells transplanted into the cerebral ventricles of neonatal and adult rats survived at least 2 mo without evidence of immunological rejection. The cells can be identified by their strong yellow fluorescent reaction with glyoxylic acid, which suggests that they maintain intact the capability of synthesizing and storing catecholamines. The cells did not show sprouting or process formation and appeared to be free in the ventricle or aggregated in clusters. This shows that cells from different animal species and from different tissue origins can be transplanted and can survive in the cerebral ventricles. When non-neoplastic tissues are transplanted between animals and man or between two animals of different species, they usually become necrotic within 6-11idays (1, 2). Normal tissue from nonembryo donors may survive a little longer if transplanted to special sites, including the anterior chamber of the eye, the brain, the cornea, the testicles, and the bone marrow space of various animal species; the cheek pouch of the hamster; and alymphatic skin flaps (1, 3). Little information is presently available on the transplantation of mammalian nervous tissue (homologous or heterologous) to brain or spinal cord (4, 5). One report has indicated evidence of rejection 2 wk after the homotransplantation of rat peripheral ganglia containing sensory neurons into the spinal cord of aniother rat with major and minor histocompatibility differences (6). By 65 days after transplantation, the neurons in this transplant have disappeared, and the ganglia are intensely infiltrated by lymphocytes. In contrast to this observation, one of us (4) has reported that homologous substantia nigra tissue from rat transplanted into the ventricles of other rats survives for at least 8-9 mo without significant signs of rejection. This data has been confirmed, and similar longlasting survival of homologous substantia nigra cells in the rat central nervous system has been reported (5). Here we report on yet another feature of cell transplants in the cerebral ventricle-the possibility of transplanting and obtaining prolonged survival of heterologous cells of neural crest origin by injecting dispersed cultured bovine adrenal medullary chromaffin cells to the cerebral ventricles of adult or neonatal rats.

RESULTS There was no alteration in the behavior or appearance of the animals. With the exception of a small area of tissue loss on the cerebral cortex at the point of needle insertion in the adult animals, there was no abnormality on gross brain examination in either neonatal or adult animals. Bright yellow fluorescent chromaffin cells were observed in the cerebral ventricles of all animals injected with the cells (Figs. 1 and 2). The cells were unique and stood out against the diffuse green fluorescence of the host brain. The appearance of the cells was similar in both neonatal and adult animals; the cells were round and without evidence of cellular processes or sprouts. The appearance of the cells was similar to that reported (8) in the tissue culture preparation on the day of the injection. The transplanted cells were usually located in the lateral ventricle and occasionally in the third ventricle. Fig. 2 shows a group of cells in the third ventricle adjacent to the dorsal portion of the medium eminence. Most of the cells appeared to be free and dispersed as individual elements in the ventricles (Fig. 1 A and C), on the ependymal surface, or on the choroid plexeses. Frequently, however, the cells were grouped as clumps within the ventricular space (Figs. 1C and 2). There was neither a difference between the appearance of transplanted cells in the neonatal and adult animals nor a difference between the cells at 10, 30, or 60 days after transplantation. Judging on the basis of the resolution of our histological method, we observed no apparent sprouting of catecholamine fluorescent structures in the brain adjacent to the ventricles. Cresyl violet staining of the same tissue sections showed an absence of any round cell infiltrate into the transplanted tissues or into the host brains adjacent to the transplanted tissues (Fig. 3).

METHODS Adult bovine adrenal medullary chromaffin cells were cultured for 1-2 wk as described (7). These cells were 95-98% pure and contained large amounts of catecholamines (25 ng/106 cells): norepinephrine 17%, epinephrine 75%, and dopamine 7%. Norepinephrine and epinephrine can be released following nicotinic receptor stimulation by a calcium-dependent mechanism (8). The cells in the culture media were centrifuged and

To whom reprint requests should be addressed at: Department of Neurology, Mount Sinai Hospital, Annenberg Bldg., Rm 14-52, 100th Street and Fifth Ave., New York, NY 10029. t Present address: Life Science Institute, Sophia University, Tokyo, Japan. *

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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FIG. 2. Chromaffm cells in the third ventricle dorsal to the medium eminence, ME. (X160.)

FIG. 1. Chromaffin cells in the lateral ventricle of an adult rat. (A) Ten days after transplantation. S, septum; C, caudate. (X47.) (B) Thirty days after transplantation. (X310.) (C) Sixty days after transplantation. (X310.)

DISCUSSION Cultured bovine adrenal medulla cells can be transplanted and maintained alive in the cerebral ventricles of an adult or neonatal rat and can survive without rejection for at least 2 mo. The prolonged survival of this cross-species transplant probably is the result of any one or all, of the following reasons: (i) the cerebral ventricles line the brain, which apparently is a relatively privileged site immunologically, and tissues transplanted to it are not readily rejected (1, 3-5, 10, 11); (ii) the transplants are small and, like other small transplants (3, 11-16), may elicit a diminished immunological response in the host; and (iii) the culturing of prospective neuroendocrine allografts prior to transplantation promotes their survival, as shown for other endocrine tissues (15, 17-22). Like other tissues, transplants into the substance of the brain require nutrition. This function is generally handled by the vascular system. The nature of the capillaries supplying these transplants seems to reflect the normal vasculature of the orthotopic tissue rather than that of the brain (23). In the case

of transplanted or metastatic tumors, the only cases thoroughly investigated, the blood-brain barrier in the substance of the tumors is absent (24-27). Since the cerebrospinal fluid is very similar in composition to blood (28, 29) and can support cellular metabolism, cells present in it, like the cells we transplanted, can remain viable without being vascularized. The absence of vascularization probably permits the blood-brain barrier to remain intact. The presence of an intact blood-brain barrier may account for the observation that transplants to the brain or anterior chamber of the eye experience a prolonged survival so long as they are not vascularized (10, 11, 30-34). Thus, we speculate that cells present in the cerebrospinal fluid are even less subject to immunological rejection than those transplanted to the already immunologically privileged brain, and may account for the success of this cross-species transplant. This contention is supported by the absence of a lymplocytic or round cell infiltrate. The chromaffin cells originate in the neural crest and have both neural and endocrine features (35-38). As such, it is conceivable that they may be used to replace a lost neural or endocrine function. When transplanted to a new environment, medullary cells can reinnervate tissues normally innervated by sympathetic fibers (35, 39, 40). Adrenal chromaffin tissue has been transplanted to the kidney capsule (41-43) and the anterior chamber of the eye (36, 39, 40, 43-48). In these new environments the adrenal chromaffin cells, like other cells of neural origin, can change their morphological characteristics (39-43, 46, 48, 49), and their synaptic relationships to other cells (41, 43, 46). This seems, however, to depend, in part upon the origin of the transplanted cells. Bovine adrenal chromaffin cells in culture, or when transplanted to the anterior chamber of the eye of a rat (K. Unsicker, personal communication), and now when transplanted to the brain of a rat, show little or no sprouting or process formation. This is in contrast to the active and extensive process formation observed when rodent or human tissue is transplanted to the anterior chamber of the eye of a rodent (39, 40, 46, 48) or cultured in vitro (35, 43, 49), or when rat tissue is transplanted to the cerebral ventricle of a rat (L. Olson, personal communication). Although the adrenal chromaffin cells do not make apparent synaptic contact, they appear to be perfectly viable in the ventricles.

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V

F

__

w

E

I

I

FIG. 3. (A) Chromaffin cells stained with glyoxylic acid 60 days after transplantation. (B) Identical tissue section as A stained with cresyl violet. V, ventricle; C, choroid plexus; E, ependyma. (X400.)

We have established that the primary culture of bovine adrenal chromaffin cells contains 75% epinephrine, 17% norepinephrine, and 7% dopamine (8). The proportions of catecholamines maintained in the cells surviving in the ventricles is not established by the present experiment. It also is difficult to establish at the present time if these cells can release these catecholamines (36, 37) upon stimulation and also if they have somatostatin (50, 51), substance P (52), enkephalins, or opiate peptides (51-56) as have been reported for fresh chromaffin tissue. Thus, it appears that it is possible to transplant isolated cells of neuroendocrine origin across species and to maintain the machinery to synthesize catecholamines for at least 2 mo. If we can demonstrate that it is possible to similarily stimulate or inhibit the release of neurotransmitters or neurohormones from these transplanted cells in vivo, then it is reasonable to consider the use of this and other primary nervous and neuroendocrine tissue culture transplants as therapeutic devices to compensate for losses in neuronal and neuroendocrine function. 1. Woodruff, M. F. A. (1960) The Transplantation of Tissues and Organs (Thomas, Springfield, IL). 2. Reematsma, K. (1968) in Human Transplantation, eds. Rapaport, F. T. & Dausset, J. (Grune and Stratton, New York), pp. 357 368. 3. Barker, C. F. & Billingham, R. E. (1977) Adv. Immunol. 25, 1-54. 4. Perlow, M. J., Freed, W. J., Hoffer, B. J., Seiger, A., Olson, L. & Wyatt, R. J. (1979) Science 204, 643-647. 5. Biorklund, A. & Stenevi, U. (1979) Brain Res. 177,555-560. 6. Zalewski, A. A., Goshgarian, H. G. & Silvers, W. K. (1978) Exp. Neurol. 59, 322-33. 7. Kumakura, K., Guidotti, A. & Costa, E. (1979) in Catecholamines: Basic and Clinical Frontiers, eds. Usdin, E., Kopin, I. J. & Barachas, J. D. (Pergamon, Oxford), pp. 61-63. 8. Kumakura, K., Karoum, F., Guidotti, A. & Costa, E. (1980) Nature (London) 283, 489-492. 9. de la Torre, J. C. & Surgeon, J. W. (1976) Histochemistry 49, 81-93. 10. Medawar, P. B. (1948) Br. J. Exp. Pathol. 29,58-69. 11. Raju, S. & Grogan, J. B. (1977) Transplant. Proc. 9, 11871191.

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