Search

Primary adrenal insufficiency is a life-threatening disorder, which requires lifelong hormone replacement therapy. Transplantation of xenogeneic adrenal cells is a potential alternative approach for the treatment of adrenal insufficiency. For a successful outcome of this replacement therapy, transplanted cells should provide adequate hormone secretion and respond to adrenal physiological stimuli. Here, we describe the generation and characterization of primary porcine adrenal spheroids capable of replacing the function of adrenal glands in vivo. Cells within the spheroids morphologically resembled adult adrenocortical cells and synthesized and secreted adrenal steroid hormones in a regulated manner. Moreover, the embedding of the spheroids in alginate led to the formation of cellular elongations of steroidogenic cells migrating centripetally towards the inner part of the slab, similar to zona Fasciculata cells in the intact organ. Finally, transplantation of adrenal spheroids in adrenalectomized SCID mice reversed the adrenal insufficiency phenotype, which significantly improved animals' survival. Overall, such adrenal models could be employed for disease modeling and drug testing, and represent the first step toward potential clinical trials in the future., (© 2023 The Authors. Xenotransplantation published by John Wiley & Sons Ltd.)

Introduction Adrenal insufficiency is a typical complication after surgical treatment of adrenal tumors, especially after the removal of both adrenal glands. Human beings are not able to survive without adrenal glands and without proper hormonal substitution. Autotransplantation of a fragment of the adrenal gland may prevent this complication. This can be done by transplanting the entire adrenal glands or its fragment, such as the adrenal cortex cells. In the case of adrenal tumors, the entire adrenal gland can not be transplanted. However, it is possible to transplant cells from the tumor-free part. Succesful adrenal autografts may result in a new treatment of adrenal insufficiency., Materials and Methods: Autograft transplantation was performed on 3 groups of Sprague Dawley rats. In the first group, physiological corticosterone concentrations were determined. These animals were not operated. In the second group, both adrenal glands were removed. Corticosterone concentrations were determined after bilateral adrenalectomy. The third group was divided into two parts. In the first subgroup, bilateral adrenalectomy was performed simultaneosly with adrenal transplant into the omentum. In the second subgroup, right adrenalectomy was performed simultaneosly with and adrenal transplant into the omentum followed a month later by left adrenalectomy. During the experiment, corticosterone concentrations were measured at 4 time points., Results: The statistical difference between corticosterone concentrations in rats after two timed adrenalectomies and rats after bilateral adrenalectomy was statistically different, but these results were far from physiological concentrations.

Patients with bilateral pheochromocytoma often require an adrenalectomy. Autotransplantation of the adrenal cortex is an alternative therapy that could potentially be performed instead of receiving glucocorticoid replacement following adrenalectomy. Adrenal cortex autotransplantation aims to avoid the side effects of long‑term steroid treatment and adrenal insufficiency. Although the function of the hypothalamo‑hypophysial system is critical for patients who have undergone adrenal cortex autotransplantation, the details of that system, with the exception of adrenocorticotropic hormone in the subjects with adrenal autotransplantation, have been overlooked for a long time. To clarify the precise effect of adrenal autotransplantation on the pituitary gland and hypothalamus, the current study examined the gene expression of hormones produced from the hypothalamus and pituitary gland. Bilateral adrenalectomy and adrenal autotransplantation were performed in 8 to 9‑week‑old male rats. The hypothalamus and pituitary tissues were collected at 4 weeks after surgery. Transcriptional regulation of hypothalamic and pituitary hormones was subsequently examined by reverse transcription‑quantitative polymerase chain reaction. Proopiomelanocortin, glycoprotein hormone α polypeptide, and thyroid stimulating hormone β were significantly elevated in the pituitary gland of autotransplanted rats when compared with sham‑operated rats. In addition, there were significant differences in the levels of corticotropin releasing hormone receptor 1 (Crhr1), Crhr2, nuclear receptor subfamily 3 group C member 1 and thyrotropin releasing hormone receptor between the sham‑operated rats and autotransplanted rats in the pituitary gland. In the hypothalamus, corticotropin releasing hormone and urocortin 2 mRNA was significantly upregulated in autotransplanted rats compared with sham‑operated rats. The authors identified significant alterations in the function of not only the hypothalamus‑pituitary‑adrenal axis, but also the adenohypophysis thyrotropes in autotransplanted rats. In the future, it will be important to examine other tissues affected by glucocorticoids following adrenal cortex autotransplantation.

Current treatment options for adrenal insufficiency are limited to corticosteroid replacement therapies. However, hormone therapy does not replicate circadian rhythms and has unpleasant side effects especially due to the failure to restore normal function of the hypothalamic-pituitary-adrenal (HPA) axis. Adrenal cell transplantation and the restoration of HPA axis function would be a feasible and useful therapeutic strategy for patients with adrenal insufficiency. We created a bioartificial adrenal with 3D cell culture conditions by encapsulation of bovine adrenocortical cells (BACs) in alginate (enBACs). We found that, compared with BACs in monolayer culture, encapsulation in alginate significantly increased the life span of BACs. Encapsulation also improved significantly both the capacity of adrenal cells for stable, long-term basal hormone release as well as the response to pituitary adrenocorticotropic hormone (ACTH) and hypothalamic luteinizing hormone-releasing hormone (LHRH) agonist, [D-Trp6]LHRH. The enBACs were transplanted into adrenalectomized, immunodeficient, and immunocompetent rats. Animals received enBACs intraperitoneally, under the kidney capsule (free cells or cells encapsulated in alginate slabs) or s.c. enclosed in oxygenating and immunoisolating βAir devices. Graft function was confirmed by the presence of cortisol in the plasma of rats. Both types of grafted encapsulated cells, explanted after 21-25 d, preserved their morphology and functional response to ACTH stimulation. In conclusion, transplantation of a bioartificial adrenal with xenogeneic cells may be a treatment option for patients with adrenocortical insufficiency and other stress-related disorders. Furthermore, this model provides a microenvironment that ensures 3D cell-cell interactions as a unique tool to investigate new insights into cell biology, differentiation, tissue organization, and homeostasis.

The occurrence of stress-inducing factors in captive crocodilians is a concern, since chronic stress can negatively affect animal health and reproduction, and hence production. Monitoring stress in wild crocodiles could also be beneficial for assessing the state of health in populations which are potentially threatened by environmental pollution. In both cases, a non-invasive approach to assess adrenocortical function as a measure of stress would be preferable, as animals are not disturbed during sample collection, and therefore sampling is feedback-free. So far, however, such a non-invasive method has not been established for any crocodilian species. As an initial step, we therefore examined the suitability of two enzyme-immunoassays, detecting faecal glucocorticoid metabolites (FGMs) with a 11β,21-diol-20-one and 5β-3α-ol-11-one structure, respectively, for monitoring stress-related physiological responses in captive Nile crocodiles (Crocodylus niloticus). An adrenocorticotropic hormone (ACTH) challenge was performed on 10 sub-adult crocodiles, resulting in an overall increase in serum corticosterone levels of 272% above the pre-injection levels 5h post-injection. Saline-treated control animals (n=8) showed an overall increase of 156% in serum corticosterone levels 5h post-administration. Faecal samples pre- and post-injection could be obtained from three of the six individually housed crocodiles, resulting in FGM concentrations 136-380% above pre-injection levels, always detected in the first sample collected post-treatment (7-15 days post-injection). FGM concentrations seem comparatively stable at ambient temperatures for up to 72 h post-defaecation. In conclusion, non-invasive hormone monitoring can be used for assessing adrenocortical function in captive Nile crocodiles based on FGM analysis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Organ preserving resection (subtotal adrenalectomy) or adrenocortical autotransplantation can preserve adrenocortical stress capacity in bilateral adrenal surgery. After adrenocortical autotransplantation approximately 30% of patients do not need exogenous steroids. Organ preserving surgery avoids steroid supplementation in more than 80% of cases. After organ preserving resections in secondary or familial diseases, however, there is a relevant risk of recurrent disease: the rate of ipsilateral recurrence in familial pheochromocytoma is approximately 20% during a follow-up of 20 years. Routine administration of exogenous steroids should be avoided after subtotal adrenalectomy as functional restitution of the residual tissue might be disturbed. Approximately 80% of patients, however, present with impaired adrenocortical stress capacity directly after surgery. Within a few weeks some 80% of patients show a sufficient functional restitution of the adrenocortical stress capacity. Organ preserving adrenal surgery should be performed endoscopically. The adrenal remnant should not be devascularized; the adrenal vein, however, can be divided without functional consequences. About one third of a normal adrenal gland usually provides sufficient adrenocortical function.

Aberrant expression of ectopic G protein-coupled receptors (GPCRs) in adrenal cortex tissue has been observed in several cases of ACTH-independent macronodular adrenal hyperplasias and adenomas associated with Cushing's syndrome. Although there is clear clinical evidence for the implication of these ectopic receptors in abnormal regulation of cortisol production, whether this aberrant GPCR expression is the cause or the consequence of the development of an adrenal hyperplasia is still an open question. To answer it, we genetically engineered primary bovine adrenocortical cells to have them express the gastric inhibitory polypeptide receptor. After transplantation of these modified cells under the renal capsule of adrenalectomized immunodeficient mice, tissues formed had their functional and histological characteristics analyzed. We observed the formation of an enlarged and hyperproliferative adenomatous adrenocortical tissue that secreted cortisol in a gastric inhibitory polypeptide-dependent manner and induced a mild Cushing's syndrome with hyperglycemia. Moreover, we show that tumor development was ACTH independent. Thus, a single genetic event, inappropriate expression of a nonmutated GPCR gene, is sufficient to initiate the complete phenotypic alterations that ultimately lead to the formation of a benign adrenocortical tumor.

Transplantation of adrenal cortical tissue may represent an alternative treatment to reestablish glucocorticoid secretion in adrenal insufficiency. In the present work, performed in adrenalectomized rats and adrenalectomized rats with a complete autotransplanted adrenal into the spleen, several hormones and biochemical parameters were measured and compared to control animals, in order to examine hormone interactions. Rats were sacrificed three weeks after surgery, and plasma and tissue samples were obtained for hormone and biochemical measurements. In adrenalectomized animals, plasma corticosterone, aldosterone and insulin levels were profoundly decreased, whereas in autotransplanted rats plasma corticosterone levels showed a partial recovery, aldosterone plasma concentrations remained low, and plasma insulin levels increased to values close to those of the controls. Both groups showed a marked elevation of plasma ACTH levels, as well as significantly increased plasma glucagon concentrations. In autotransplanted animals, most of the biochemical parameters, which were altered in adrenalectomized rats, returned to normal levels. These results suggest that increased glucagon levels in adrenalectomized and autotransplanted animals, may contribute to the marked increase of plasma ACTH, and could also be important in the recovery of plasma glucose and hepatic glycogen observed in autografted rats. Since high glucagon concentrations alone were unable to normalize carbohydrate levels in adrenalectomized animals, it appears that glucagon can act only in the presence of corticosterone.

Purpose: Autotransplantation of the adrenal cortex may be a therapeutic alternative in the future. For successful adrenal transplantation revascularization is necessary. It is possible that vascular endothelial growth factor (VEGF), which is a potent angiogenic peptide, may have some roles in adrenal transplantation through 2 its receptors, kinase insert domain-containing region (Flk-1) and fms-like tyrosine kinase (Flt-1). Therefore, we studied sequential changes in expression of VEGF, Flk-1 and Flt-1 in regenerated adrenal., Materials and Methods: Eight to 9-week-old male Wistar rats underwent bilateral adrenalectomy and immediate adrenal capsular autotransplantation. The expression of VEGF, Flk-1 and Flt-1 was analyzed by immunohistochemistry and reverse-transcriptase-polymerase chain reaction., Results: Angiogenesis was observed in the remodeling of adrenal sinusoidal endothelium during adrenal regeneration. Reverse transcriptase-polymerase chain reaction and immunohistochemistry showed that VEGF expression increased in grafted tissue with time after transplantation and its Flk-1 receptor, which localized to endothelial cells, increased transiently during the regeneration process. Immunostaining for Flt-1 receptor was identified in adrenocortical cells and its intensity gradually increased during adrenal regeneration., Conclusions: During adrenal gland regeneration VEGF and its receptors Flk-1 and Flt-1 are thought to be involved in neovascularization.

Adrenocortical cell transplantation is a powerful technique for the investigation of the regulation of adrenocortical structure and function. Some classical organ and tissue transplantation experiments suggest that the success of transplantation depends on the activity of the pituitary gland and other endocrine systems, and is therefore influenced by the host animals' own adrenal glands. For this reason, our experiments have usually been performed on adrenalectomized animals. However, we show here that cell transplantation experiments, involving the introduction of bovine adrenocortical cells into scid mice, do produce transplant tissues in the presence of the host animals' adrenal glands. However, the tissue that forms is small and its cells also smaller than usual. When the adrenals of such animals are removed in a second surgical procedure, the transplants show a rapid increase in steroidogenic function and a slower increase in size, over several weeks. We conclude that the initial process by which transplanted adrenocortical cells organize into a tissue structure is not affected by the presence of the host animals' adrenal glands, but the growth of the transplants is limited until the adrenal glands are removed.

Background: Hormone substitution for the treatment of adrenocortical insufficiency does not adequately substitute the physiologic circadian secretion of corticosteroids and leads to long-term sequelae and reduced quality of life. The lack of adaptation to physical and psychologic stress situations may lead to life-threatening Addison's crises. Allogeneic transplantation of adrenal cortex could offer an intriguing alternative. Adrenocortical grafts were demonstrated to proliferate and produce corticosteroids in physiologic concentrations after transplantation., Methods: K -transgenic murine lymphocytes and allogeneic adrenal cortex cells were cocultured in mixed lymphocyte reactions to examine the alloimmune response; lymphocytes from T-cell receptor transgenic mice and normal mice, respectively, served as responder cells. The effects of corticosteroids secreted by adrenocortical cells were antagonized by the steroid receptor antagonist mifepristone, whereas the impact of cell-cell interactions was differentiated with transwell culture systems., Results: Coculture of adrenal cortex cells in mixed lymphocyte reactions markedly suppressed lymphocyte proliferation. Transwell cultures demonstrated that adrenocortical cells exerted their effects by a soluble factor that was only partially antagonized by mifepristone., Conclusion: In vitro, the presence of adrenocortical cells potently suppressed allogeneic immune responses. This effect was not exclusively the result of the secretion of corticosteroids, indicating an additional immunomodulatory property of adrenocortical cells.

Cell transplantation provides a way to study genes that may be important in human tissue aging. Studies on gene action in human cells are usually restricted to cell culture investigations and clinical observations. Differences in human and rodent cellular biology, particularly with respect to telomere dynamics, show the need for new systems for investigating aging that use human cells or cells of other large, long-lived mammals, such as bovine cells. The system we describe uses human and bovine adrenocortical cells transplanted into scid (severe combined immunodeficiency) mice. They form a vascularized tissue structure that can replace the essential functions of the animals' own adrenal glands. The cells may be genetically modified before introduction into the animal. Using hTERT (telomerase reverse transcriptase) and oncoproteins, we show the potential for investigating gene action in genetically modified tissues created by cell transplantation.

Expression of TERT, the reverse transcriptase component of telomerase, is necessary to convert normal human cells to cancer cells. Despite this, "telomerization" by hTERT does not appear to alter the normal properties of cells. In a cell transplantation model in which bovine adrenocortical cells form vascularized tissue structures beneath the kidney capsule in scid mice, telomerization does not perturb the functional tissue-forming capacity of the cells. This cell transplantation model was used to study the cooperation of hTERT with SV40 T antigen (SV40 TAg) and oncogenic Ras in tumorigenesis. Only cells expressing all three genes were tumorigenic; this required large T, but not small t, antigen. These cells produced a continuously expanding tissue mass; they were invasive with respect to adjacent organs and eventually destroyed the kidney. Cells expressing only hTERT or only Ras produced minimally altered tissues. In contrast, SV40 TAg alone produced noninvasive nodules beneath the kidney capsule that had high proliferation rates balanced by high rates of apoptosis. The use of cell transplantation techniques in a cell type that is able to form tissue structures with or without full neoplastic conversion allows the phenotypes produced by individual cooperating oncogenes to be observed.

Intradermal, as opposed to subcutaneous, cell transplantation was previously shown to be advantageous for tumor cell growth, but this site has not been used for transplantation of normal nonneoplastic cells. In preliminary experiments we found that it was difficult to control the size and shape of transplants when we injected dissociated cells intradermally. This problem was solved by placing cells in nongelled, pepsin-solubilized collagen prior to injection. This technique permitted the successful transplantation of normal bovine adrenocortical cells and of neoplastic cells (3T3 cells secreting FGF) in scid mice. Primary bovine adrenocortical cells formed functional vascularized tissue and the transplants rescued the animals from the lethal effects of adrenalectomy. The histological structure of transplant tissues resembled that previously observed when cells were transplanted in the subrenal capsule space. We also used a line of 3T3 cells that has been genetically modified to secrete a form of acidic FGF. When transplanted intradermally in collagen, they formed rapidly enlarging masses of cells that could easily be palpated beneath the skin of the animal. Intradermal injection of cells in pepsin-solubilized collagen is a simple and reliable technique for transplanting normal primary cells and preneoplastic cells. The ability to grow both types of cells in an easily accessible site allows less invasive monitoring of growth, angiogenesis, and other features of the transplant.

To establish an experimental model for replacement of endocrine organ function by xenotransplantation, human adrenocortical cells from postnatal donors were transplanted beneath the kidney capsule of adrenalectomized scidmice together with mitomycin C-treated 3T3 cells that secrete FGF. Adrenocortical cells from seven donors, male and female, ranging from 6-50 years of age, were used. 12 of 13 animals survived > 16 days following surgery. After 50 days they were sacrified to allow assessment of the histology and ultrastructure of tissue formed from the transplanted cells. Only 1 of 23 adrenalectomized sham-operated animals survived > 16 days. In all surviving animals, vascularized adrenocortical tissue formed at the site of transplantation. Cortisol, the normal human glucocorticoid, was present in the plasma of these animals, replacing corticosterone, the mouse glucocorticoid. Some animals, but not most, had measurable aldosterone. The tissue formed from the transplanted cells showed histological and ultrastructural features of normal adrenal cortex. Mitochondria had tubulo-vesicular cristae and there were prominent microvilli between cells. Tissues had a well-developed vasculature, sometimes with large sinusoidal vessels. Proliferation in the transplant tissues was very low. These results show that tissue formed from transplanted human adrenocortical cells is able to replace the essential functions of the adrenal gland in scid mice. This demonstrates that transplanted human endocrine cells can functionally replace a surgically removed endocrine organ in a host animal.

Laparoscopic surgery for urological conditions has now become popular worldwide. The case is reported of a 56-year-old woman who underwent simultaneous bilateral laparoscopic adrenalectomy for adrenocorticotropic hormone-independent macronodular adrenocortical hyperplasia (AIMAH), followed by autotransplantation of resected adrenal gland fragments. Simultaneous laparoscopic adrenalectomies seem feasible for a patient with AIMAH because of its minimally invasive nature. However, autotransplantation of adrenal fragments failed in this patient with AIMAH.

Cell transplantation provides a way to compare the regulation of cell proliferation in the same cell type in cell culture and in a vascularized tissue structure in a host animal. The cyclin-dependent kinase inhibitors p57(KIP2), p21(WAF1/CIP1/SDI1) and p27(KIP1) have been extensively studied in cell culture but their role in growth control in tissues is less well understood. In the present experiments we compared the behavior of cell cycle inhibitors in human and bovine adrenocortical cells in culture and following cell transplantation in scid mice. p57 was expressed in the majority of cells in the intact human adrenal cortex. However, double immunofluorescence showed that cells that are in the cell cycle are p57(-) adrenocortical cells, p57 and p27 levels were not affected by inhibition of growth at high cell density, whereas p21 was higher in dividing than growth-inhibited cells. However, p21 was also high in senescent adrenocortical cells. After transplantation of human adrenocortical cells in scid mice, p57 and p27 were observed in most cells in the transplant tissue. Over time the number of p21(+) cells decreased greatly in human adrenocortical cells, but not in bovine adrenocortical cells. This difference correlated with lower levels of cell division (assessed by Ki-67 or incorporation of bromodeoxyuridine) in the human cells in transplant tissues in comparison to bovine cells. The differences between human and bovine cells were observed both when cells were transplanted beneath the kidney capsule and when cells were injected subcutaneously in collagen gel. We conclude that the behavior of p57, but not p21, is consistent with a role as a physiological mediator of proliferative quiescence in the adrenal cortex. The high level of p21 in dividing adrenocortical cells in culture, and in bovine adrenocortical cells in transplant tissues, may be a response to conflicting positive and negative growth influences. Cells may enter the cell cycle under the influence of a strong positive mitogenic signal, but coexisting negative growth stimuli trigger a p21-dependent block to further progression through the cell cycle. This model suggests that bovine adrenocortical cells respond to positive growth stimuli in transplant tissues but human cells lack this response., (Copyright 2001 Academic Press.)

This study tested the possibility of adrenal autotransplantation in rats. Since the cortex and the medulla of the adrenal gland were from different origin embryologically, either whole adrenal glands (ADR), or capsule and cortex (CAP) or medulla (MED) were autotransplanted in the subcutaneous tissue. The functions of regenerated adrenal nodules were tested by measuring plasma corticosterone levels every fortnight. At the end of 9 weeks the rats were exposed to hypovolemic shock followed by naloxone injection to reverse the shock response. Results showed that rats transplanted with either cortex or whole adrenal started secreting corticosterone at 5 weeks post-transplantation (107.73 +/- 21.98 ng/ml, 126.04 +/- 48.41 ng/ml, respectively). Corticosterone levels increased to the value which were not significantly different from control by 9 weeks post-transplantation. However, rats transplanted with adrenal medulla showed very low corticosterone levels. Nine weeks post-transplantation, the mean blood pressure (MBP) of the CAP group was 135 +/- 13 mmHg and was not significantly different from sham-operated controls, whereas MBP of MED group was significantly lower than sham-operated animals (99 +/- 11 mmHg versus 141 +/- 9 mmHg). The MBP of the ADR group was also lower compared to sham-operated controls (112 +/- 17 mmHg P < 0.05). The MBP of the adrenal group was not statistically significant compared to the CAP group. After 1% body weight haemorrhage, the MBP decreased significantly in ADR (45 +/- 5 mmHg, P < 0.05) and MED group (36 +/- 9 mmHg, P < 0.001) compared to sham-operated rats (78 +/- 11 mmHg) but not in the CAP (56 +/- 9 mmHg). It was concluded that autotransplanted whole adrenal or adrenocortical tissues survived subcutaneously and produced sufficient corticosterone to alleviate haemorrhagic shock. Adrenal medullary tissue failed to regenerate subcutaneously and the presence of adrenal medullary tissue may suppressed the growth of transplanted adrenal gland.

Xenotransplanted adrenocortical tissue of clonal origin was formed in immunodeficient (scid) mice by using techniques of cell transplantation. The experiments reported here used a single clone of bovine adrenocortical cells, but 5 of 20 other randomly selected clones also formed tissue. Most adrenalectomized animals bearing transplanted cells survived indefinitely, demonstrating that the cells restored the animals' capacity to survive in the absence of sodium supplementation. Formation of well-vascularized tissue at the site of transplantation was associated with stable levels of cortisol in the blood, replacing the mouse glucocorticoid (corticosterone). Ultrastructurally, the cultured cells before transplantation had characteristics of rapidly growing cells, but tissue formed in vivo showed features associated with active steroidogenesis. These experiments show that an endocrine tissue can be derived from a single, normal somatic cell.

Surgical strategies for pheochromocytomas in patients with multiple endocrine neoplasia (MEN) type 2 syndrome have been controversial. The purpose of this study is to review the current status of patients with MEN 2 who underwent adrenalectomy with or without adrenal autotransplantation. We studied 15 patients with MEN 2A who underwent adrenal surgery between 1981 and 1992. The follow-up survey included physical examination and biochemical determinations. The median period from initial surgery to follow-up was 54 months (range, 0-145 months). Initial bilateral total adrenalectomy was performed on seven patients, and subtotal adrenalectomy was carried out on two. Among six patients who initially underwent unilateral adrenalectomy, four had remained normotensive (median follow-up, 61 months), whereas the other two patients had to undergo reoperation on the contralateral side because of recurrent symptoms. Two patients were suspected of having had a relapse of the disease after total adrenalectomy. Seven patients underwent adrenal autotransplantation; however, none of them were able to discontinue glucocorticoid replacement therapy. In MEN 2A patients having large pheochromocytomas on only one side, unilateral adrenalectomy can be a suitable alternative to bilateral adrenalectomy in terms of blood pressure control and preservation of adrenocortical function. The attempt to preserve adrenocortical function by autotransplantation is discouraged.

The transplantation of cells that secrete neuroactive substances with analgesic properties into the CNS is a novel method that challenges current approaches in treating chronic pain. This review covers pre-clinical and clinical studies from both allogeneic and xenogeneic sources. One cell source that has been utilized successfully is the adrenal chromaffin cell, since such cells constitutively release catecholamines, opioid peptides, and neurotrophic factors; release can be augmented with nicotine. Other graft sources include AtT-20 and B-16 cell lines which release enkephalins and catecholamines, respectively. For grafting in rodents, adrenal medullary tissue pieces are transplanted to the subarachnoid space. Chromaffin cell transplants can decrease pain sensitivity in normal rats using standard acute pain tests (paw-pinch, hot-plate, and tail-flick). In addition, transplants can restore normal pain thresholds in rodent models of chronic pain (formalin, adjuvant-induced arthritis, and sciatic-nerve tie) which closely similate the pathologies of human chronic pain conditions. Xenografts have been studied due to concerns that future application for human pain may be limited by donor availability. Despite immune privileges of the CNS, xenografts require at least short-term immunosuppression to obtain a viable graft. Cell encapsulation is one method of sustaining a xenograft (in rat and human hosts) while circumventing the need for immunosuppression. Clinical studies have been initiated for terminal cancer patients with promising results as assessed by markedly reduced narcotic intake, visual analog scale ratings, and increased CSF levels of catecholamines and met-enkephalin.

The proliferative activity, the organization and the corticosterone expression of adrenocortical cells in an experimental adrenal regeneration process after the transplantation of neonatal adrenal glands to adult hosts was investigated. Three days after transplantation, the medullar and the innermost adrenocortical cells of the neonatal adrenal glands showed degenerative and necrotic changes due to the lack of vascular supply. The remaining outermost adrenocortical cells did not display any PCNA immunoreaction. The first PCNA expression, pointing out the beginning of the proliferative cycle, was observed in a 45.4% of the adrenocortical cells, one week after transplantation. After three weeks, several regenerated adrenocortical nodules with a bigger size than the one observed in the previous periods were seen. In these nodules, while the outermost adrenocortical cells were disposed in parallel to the capsule or in rounded groups, the bulk of the regenerated mass width was composed of cells forming longitudinal cords. PCNA immunoreaction was almost exclusively restricted to subcapsular cells (62.5%) and to cells of the outermost portion of the cords (32.5%), the global percentage of PCNA immunopositive cells being 18.4%. Twelve weeks after transplantation, regenerated adrenocortical cells were arranged in three layers: glomerulosa, fasciculata and reticularis. Only 1.85% of the adrenocortical cells were PCNA immunopositive. Although in the early stages of the regeneration process, all the adrenocortical cells, both proliferating and non proliferating cells expressed corticosterone, a restriction of this immunoreactivity to the zonae fasciculata and reticularis was observed when cell zonation was apparent.

Substance P (SP) increased aldosterone secretion of rat adrenal slices, but not of isolated zona glomerulosa cells, and this effect was annulled by two specific antagonist of SP (SP-A). Both tissue preparations displayed an aldosterone secretory response to isoprenaline (IP) that was blocked by l-alprenolol (AL). AL reversed the aldosterone response of adrenal slices to IP, SP, or IP plus SP, whereas SP-A only suppressed that to SP. Quarters of adrenocortical autotransplants, which are completely deprived of chromaffin cells, showed an aldosterone response to IP, but not to SP. These findings suggest that the mechanism underlying the aldosterone secretagogue action of SP probably involves the stimulation of catecholamine release by adrenal medulla chromaffin cells.

Objective: Evaluate long-term efficacy of autologous adrenal-to-caudate transplants in idiopathic Parkinson's disease refractory to medical treatment., Design: Subjects underwent evaluations several times preoperatively on the University of California-Los Angeles Parkinson's Disease Disability Scale and the Hoehn and Yahr stage of disease. Postoperatively, they were also repeatedly rated on the Unified Parkinson's Disease Rating Scale., Setting: Clinical visits and surgery took place at the University of California-Los Angeles Center for the Health Sciences., Patients: Three men and one woman, ages 44 to 55 years, were followed up for several years preoperatively. At surgery, disease durations ranged from 7 to 16 years. Originally, all patients had a good response to levodopa, but for several years preoperatively, they had had fluctuating responses and a short duration of drug action., Intervention: Right adrenalectomy was performed through a midline abdominal incision. Open craniotomy exposed the head of the right caudate into which pieces of adrenal medulla, 1 to 2 mm in size, were implanted., Main Outcome Measures: Scores on the three major scales (see "Design") were augmented with the number of hours "off" per day and severity of abnormal involuntary movements. Disease progression of each patient was compared with his own preoperative course and with those of a cohort of patients with Parkinson's disease followed up for 14 years who had received medical treatment without transplant surgery., Results: After 4 years, transplants continued to be beneficial to three patients and had been of brief transient benefit to the fourth. The course of disease was more benign postoperatively than preoperatively and was more slowly progressive than that in the cohort., Conclusion: Improvement was not sufficient to justify adrenal transplants as routine therapy but does point the way to the use of other dopamine tissue transplantation.

The effect of SRIF and its antagonist cyclo(7-aminoheptanonyl-Phe-D-Trp-Lys-Thr magnitude of Bzl)(SRIF-A) were studied in sham-operated and bilaterally adrenalectomized rats bearing ACTH- and angiotensin II (ANG-II)-responsive adrenocortical autotransplants. SRIF-A (10(-5) M) completely annulled SRIF (10(-6) M)-induced inhibition of ANG-II (10(-8) M)-evoked rise in aldosterone (ALDO) secretion by both dispersed zona glomerulosa (ZG) cells and autotransplant slices. A 7-day intraperitoneal infusion with SRIF (0.3 nmol.kg-1.min-1) significantly lowered plasma ALDO concentration (PAC) in both groups of animals, without affecting plasma renin activity and the plasma levels of ACTH and corticosterone. This treatment caused a marked atrophy of adrenal ZG and its parenchymal cells (without inducing any significant change in the zona fasciculata morphology), as well as of ZG-like cells of autotransplants. Isolated ZG cells and autotransplant slices from SRIF-infused rats evidenced a notable decrease in both their basal and maximally ACTH- or ANG-II-stimulated ALDO production. The simultaneous infusion of rats with SRIF-A (3 nmol.kg-1.min-1) completely reversed all these effects of SRIF. The prolonged infusion with SRIF-A alone caused, in sham-operated rats, a marked increase in PAC and a significant hypertrophy of ZG and ZG cells; basal and maximally-stimulated ALDO secretion of dispersed ZG cells was also notably raised. Conversely, SRIF-A infusion did not evoke any appreciable effect in autotransplanted rats. These findings suggest that endogenous SRIF is specifically involved in the negative control of the secretion and growth of the rat adrenal ZG. Since regenerated adrenocortical autotransplants, which are responsive to SRIF but not to SRIF-A infusion, are completely deprived of chromaffin cells, the hypothesis is advanced that adrenal zona medullaris may be the source of endogenous SRIF regulating ZG function.

Adrenal autotransplantation after bilateral total adrenalectomy has been utilized to eliminate the need for replacement therapy and to prevent the late occurrence of Nelson's syndrome in some patients with Cushing's disease. It is possible to follow these cases up closely today, owing to the highly developed hormonal evaluation and imaging techniques. In this study, two patients who underwent bilateral total adrenalectomy and cortex autotransplantation are presented. The autografts were found functional and the patients had not required any steroid replacement therapy.

We have used a polyclonal antibody to the biosynthetic adrenocortical enzyme, 3-beta-hydroxysteroid dehydrogenase, to demonstrate rat adrenal cortical tissue in situ, in vitro, and in intracerebral adrenal suspension grafts. This technique provides a means of identifying the degree to which adrenal medulla grafts are in practice contaminated by adrenal cortical cells and allows analysis of the extent to which the presence of cortical cells influences the viability, differentiation, and functional efficacy of intracerebral adrenal grafts.

Well documented reports of the successful transplantation of human adrenal cortical tissue cannot be found in the literature. In 1951 we achieved the successful transplantation of human embryonic adrenal gland (cortical tissue) in a patient with symptomatic adrenal insufficiency (Addison's disease), apparently the first instance of histologically documented successful homografting of human adrenal cortex. Because of its historical pertinence, the authors, many years later, herein report on this case, which appeared in the senior author's medical thesis. The report must be viewed in the context of the existing clinical knowledge and technology available 40 years ago.

A morphological and autoradiographic study was made of the adrenal gland of adult male rats after autotransplantation. The simple technique involved placement of pieces of the adrenal gland in a dorsal plane between the skin and muscle. Animals for morphological studies were sacrificed at 2, 3, 4, 7, 15, 30, 90, and 180 days after autotransplantation. Those for autoradiographic studies were sacrificed at 2, 3, 7, and 15 days after autotransplantation, with 3H-thymidine being administered intraperitoneally 2 h before sacrifice. Sham-operated animals were used as controls. The majority of glandular adrenal cells suffered necrosis in the first days (2 and 3) after autotransplantation. Up until 15 days and after revascularization, morphological features of the cells were compatible with protein synthesis exhibiting a developed RER, scarce SER, and mitochondria with tubular and lamellar cristae. These data may correspond to a proliferative phase of glandular cells. At day 15, cells showed morphological signs of steroidogenic activity (mitochondria with vesicular cristae, increase of SER), and at day 30, an increased number of microvilli were seen. Between 30 and 90 days zonation of the adrenal was evident with glomerulosa, fasciculata, and reticular zones readily apparent. The quantitative analysis showed a significant increase of the volumetric density of mitochondria and microvilli between the days 7 and 30. Autoradiographic studies showed an intense labelling of fibroblast-like cells at days 2 and 3 and of glandular cells at days 7 and 15, which was confirmed by the quantitative studies. Corticosterone in autotransplanted animals decreased during the first 15 days, but after 30 days the values were similar to controls. The model reported here seems to be good for study of the regeneration of the adrenal gland and can be a simple, useful, and reproducible method for adrenal transplantation.

Microencapsulation of adrenal cells is proposed for reducing the nonspecific inflammatory reaction observed around polymer implants. This hypothesis was tested by comparing both host cellular reaction and the surrounding graft cell populations which appeared either when agarose embedded cells or when empty agarose beads were implanted. Our results showed that the fibrotic material that surrounded the implanted empty agarose microbeads was not as severe and important when adrenal cells were present. Similarly, T lymphocyte population surrounding the graft was considerably reduced together with the percentage of CD4 and CD8 positive cell subpopulations. The activation macrophage marker IaD disappeared. Our results support the hypothesis that embedded adrenal cells may be a suitable solution for reducing early inflammatory events due to microcapsules implantation.

Regenerated adrenocortical nodules were obtained by implanting fragments of the capsular tissue of excised adrenal glands into the musculus gracilis of rats (Belloni et al. 1990). Five months after the operation, operated rats showed a normal basal blood level of corticosterone, but a very low concentration of circulating aldosterone associated with a slightly increased plasma renin activity (PRA). Regenerated nodules were well encapsulated and some septa extended into the parenchyma from the connective-tissue capsule. The majority of parenchymal cells were similar to those of the zonae fasciculata and reticularis of the normal adrenal gland, while zona glomerulosa-like cells were exclusively located around septa (juxta-septal zone; JZ). In vitro studies demonstrated that nodules were functioning as far as glucocorticoid production was concerned, while mineralocorticoid yield was very low. Prolonged sodium restriction significantly increased PRA and plasma aldosterone concentration, and provoked a marked hypertrophy of JZ, which was due to increases in both the number and average volume of JZ cells. Accordingly, the in vitro basal production of aldosterone and other 18-hydroxylated steroids was notably enhanced. The plasma level of corticosterone, as well as zona fasciculata/reticularis-like cells and in vitro production of glucocorticoids by regenerated nodules were not affected. These findings, indicating that autotransplanted adrenocortical nodules respond to a prolonged sodium restriction similar to the normal adrenal glands, suggest that the relative deficit in mineralocorticoid production is not due to an intrinsic defect of the zona glomerulosa-like JZ, but is probably caused by the impairment of its adequate stimulation under basal conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Regenerated adrenocortical nodules were obtained by implanting in the musculus gracilis of rats fragments of the capsular tissue of their excised adrenal glands. Five months after operation, transplanted rats showed a slightly elevated blood concentration of adrenocorticotropin (ACTH), a moderately reduced plasma level of corticosterone (PBC) and a very low concentration of circulating aldosterone (PAC). Regenerated nodules were well encapsulated, and from the connective capsule some septa dipped into the parenchyma. Subcapsular-outer (OZ) and inner (IZ) cells were similar to those of the zona fasciculata/zona reticularis (ZF/ZR) of the normal gland; juxta-septal (JZ) cells resembled those of the zona glomerulosa (ZG). Prolonged (14 days) ACTH infusion normalized PBC and caused a conspicuous hypertrophy of transplanted tissue, which was coupled with a marked hypertrophy of ZF/ZR-like OZ and IZ cells and a notable rise in the basal in vitro production of corticosterone. Conversely, ACTH infusion strikingly lowered PAC, reduced the number of ZG-like JZ cells, and decreased both basal and stimulated secretion of 18-hydroxylated steroids by transplants in vitro.

This paper describes the function and morphology of regenerated adrenocortical nodules obtained by implanting, in the musculus gracilis of rats, several (n = 6-7) fragments of the capsular tissue of their excised adrenal glands. Four months after the operation, each bilaterally adrenalectomized rat developed six or seven well encapsulated adrenocortical nodules about 2-3 mm in diameter and always lacking chromaffin cells, and displayed almost complete normalization of basal and stimulated blood levels of corticosterone, but not of aldosterone. In vitro study showed that regenerated nodules were well functioning as far as glucocorticoid production was concerned. Accordingly, electron microscopy and stereology indicated that the majority of the parenchymal cells (independently of their location in the outer subcapsular, middle, or inner portions) closely resembled those of the zonae fasciculata/reticularis of the adrenal gland of age-matched sham-operated rats. By contrast, regenerated nodules evidenced a relative impairment in aldosterone secretion, and this was coupled with the presence of only a few zona glomerulosa-like cells. Such cells were grouped in small islets located near the few connective trabeculae detaching from the capsule, and autoradiography showed that they were the only parenchymal elements of the nodule able to bind [125I]angiotensin-II. The possibility is suggested that the paucity of zona glomerulosa-like cells in regenerated nodules could be ascribed to the absence of zona medullaris, which is currently thought to exert a paracrine control on the growth and secretion of zona glomerulosa in the rat adrenal glands.

The factors involved in regeneration of the autotrasplanted adrenal cortex were studied experimentally in dogs. On the basis of a number of conditions necessary for the regeneration of adrenocortical tissue, the dogs were divided into 4 groups according to differences in substitution therapy following autotransplantation. The only group to demonstrate long-term survival received an intermittent substitution of cortisone acetate after the completion of 6 days postoperative replacement therapy. The hormonal functions after adrenocortical autotransplantation were examined in the long-term survivers. The secretory function of adrenocortical tissue after autotransplantation was confirmed by the detection of serum concentrations of aldosterone and cortisol, however, estrogens were not detected in the peripheral blood stream following autotransplantation. The negative response to the synthetic ACTH stimulation test observed in this study was evidence of the functional limitation of regenerated cortical tissue. Clinically, however, the procedure performed in this study will be beneficial for patients with advanced and metastatic breast cancer, not only as a form of surgical hormonal therapy, but also as an effective palliative method of treatment.

In autotransplants of 1st group which exhibited all ultrastructural features of steroidogenesis most of cortical blood vessels were characterized by normal morphology corresponding to the vessels of control adrenal glands. However, 2nd group in which most of autotransplants showed possibility of steroid hormones synthesis capillaries were of changed structure. High endothelial cells did not form fenestrated membranes and most frequently they were situated on basement membrane composed of several layers. In autotransplants of 3rd group, devoid of steroidogenic features, all cortical vessels possessed changed structure. High endothelial cells did not form fenestrated membranes and they were situated on very thickened uniform or multi-layered basement membranes.

Life-long hormone replacement therapy is today the only form of therapy for several endocrine insufficiencies. Transplantation of endocrine tissues could represent a more physiological approach to the treatment of different syndromes such as adrenal insufficiency, diabetes or hypoparathyroidism. Successful experimental islet transplantation across major histocompatibility barriers using cultural pretreatment of the insulin producing tissue in conjunction with temporary immunosuppression of the recipients, encouraged us to attempt similar approaches with parathyroids, adrenal cortex and adrenal medulla. Either bilateral parathyroidectomy or bilateral adrenalectomy were performed on the recipient rats before transplantation, followed by the implantation of parathyroid, adrenal cortical or adrenal medullary tissue beneath the renal capsule. The tissue was transplanted either immediately after its dissection or after pretreatment with 1 week culture. Some of the recipients received a short term cyclosporine treatment. The results indicated that parathyroid and adrenal medulla transplants were often infiltrated by mononuclear cells 30 days after transplantation. In contrast, adrenal cortical tissue was well preserved 4 weeks after transplantation, without showing any sign of rejection when a combination of pre-transplant culture of the adrenal tissue and temporary cyclosporine treatment of the recipients was used.

Cushing's disease has come full cycle. As originally asserted more than 50 years ago, modern diagnostic techniques now demonstrate an adrenocorticotropic hormone (ACTH) secreting pituitary adenoma in approximately 80% of such patients. At this historical juncture, we report a long-term follow-up of our 17 patients who underwent adrenalectomy (8) or later adrenalectomy plus adrenal autotransplantation (9) between 1955 and 1976. Two patients died soon after surgery and five others died later of "natural" causes. Four others moved away but were stable when last contacted. Of the six patients who remain available for current follow-up, three have undergone hypophyseal surgery. Another patient has evidence of pituitary enlargement, and the remaining two are yet to undergo computerized tomography (CT) scan. Four illustrative cases are reviewed in some detail. One case presented with Nelson's syndrome and acute onset blindness. The second represented multiple endocrine adenomatosis with hyperparathyroidism in addition to Cushing's disease. The third exhibited Cushing's syndrome from the autotransplants, finally cured by hypophysectomy. The fourth exhibited huge ACTH levels from a large pituitary adenoma that could not be totally resected and recurrent Cushing's syndrome associated with large autotransplant "adenomas." The initial surgical treatment of choice is pituitary adenectomy. Bilateral adrenalectomy will remain useful where curative pituitary surgery is not feasible. Neither pituitary irradiation nor medical therapy has been truly effective in our patients. Adrenal autotransplants survive, to some extent, in virtually all patients. However, the degree of function is variable, and the full function may not be achieved for many months or even years. Functioning autotransplants have not prevented Nelson's syndrome, and they would appear to offer little practical benefit at this time.

56 cases of adrenalectomy and autotransplantation of human adrenal tissue have been reported in the literature since 1955. The most common indication was bilateral adrenal hyperplasia. A reduction in postoperative corticosteroid substitution and a return to almost physiological function on discontinuation of long-term substitution can be achieved. Recurrent disease is easily cured by partial excision of the transplanted tissue, situated usually in small muscle pockets, under local anaesthesia. The loss of adaptation to circadian rhythm and stress are adverse effects. Further development of this technique, which is still relatively rarely implemented, may make it more universally applicable for a wider indication of bilateral adrenalectomy, without loss of adrenal function.

Thymusaplastic mutants of mice (nu/nu) and rats (rnu/rnu) were used as recipients for tissue of one aldosteronoma of a 21-year-old male patient with Conn's syndrome and for tissue of one adenoma of a 28-year-old female patient with Cushing's syndrome. In this animal experimental study we succeeded in demonstrating that congenitally athymic rodents are ideal recipients for xenotransplantation of adrenal cortical tissue. The graft viability was demonstrated by obtaining light and electron micrographs of the biopsied transplants and its endocrine function documented by the measurements of plasma renin, cortisol, and urinary excretion of aldosterone, cortisol, and corticosterone. Fifteen patients are presented who were surgically treated for Cushing's disease. Fourteen patients with adrenocortical hyperplasia underwent total adrenalectomy and two had after adrenalectomy autotransplantation of adrenal tissue in the forearm muscle. One patient was treated with a transsphenoidal, partial hypophysectomy. Our clinical experience with Cushing's disease showed that the initial surgical procedure may be directed to the pituitary and only in unsuccessful surgery to the adrenals. Adrenal autotransplantation after total adrenalectomy is an obsolete procedure.

Autotransplantation of human adrenal tissue has been attempted often, but results have been difficult to evaluate and success has been infrequent. Factors that may affect success include: volume of transplanted tissue, recipient site, inclusion of cortical capsule with the autograft, systemic or local growth factors, and the timing of evaluation. We have evaluated a model of autotransplantation in rats that will permit examination of these factors. Male Sprague-Dawley rats were assigned to (1) bilateral adrenalectomy (ADX), (2) bilateral adrenalectomy with immediate autotransplantation to a flank muscle pocket of one third of a single adrenal gland with its capsule attached (TX), or (3) sham operation. Animals were provided with 0.9% saline solution ad lib. At 2, 4, 6, and 12 weeks after surgery animals were stressed by brief exposure to ether and 15 minutes later had blood collected for determination of corticosterone concentration (C). ADX animals consistently weighed less than either TX or sham-operated animals; weights of sham-operated and TX animals were similar. Sham-operated animals uniformly had C levels higher than ADX or TX animals. At 2 and 4 weeks after surgery, C was similar in ADX and TX; but at 6 and 12 weeks, TX animals had higher values than had ADX animals. With this model, graft function can be demonstrated at 2 weeks by comparing body weight and at 6 weeks by comparing postether levels of C in TX animals to ADX animals. ADX animals can be maintained without steroid replacement on a regular diet with 0.9% saline solution ad lib. This model will permit examination of technical and physiologic influences on transplant success with both fresh and cryopreserved tissue and may lend itself to radionuclide or nuclear magnetic resonance assessment of graft function.

The regeneration of adrenocortical autotransplants in the rat spleen has been investigated by light and electron microscopy. Up to the seventh day after implantation, adrenal grafts showed large areas of necrosis and contained many degenerating (apoptotic) adrenocortical cells, some mesenchyme-like poorly differentiated elements, and occasional viable parenchymal cells. These last cells possessed mitochondria with scanty tubular cristae and few profiles of smooth endoplasmic reticulum. After 15 days of regeneration, adrenal grafts were reduced in volume, but contained only viable adrenocortical cells; after 30-36 days, autotransplants were noticeably enlarged and surrounded by an evident connective tissue capsule. Regeneration was closely associated with the morphological differentiation of adrenocortical cells, which one month after transplantation were found to assume all the typical features of adult rat zona fasciculata elements (i.e. mitochondria with vesicular cristae, abundant smooth endoplasmic reticulum, some lipid droplets and a well developed Golgi apparatus).

Adrenal medullary tissue from Wistar-Furth rats was transplanted beneath the renal capsule of Lewis rats to determine the effect of in vitro culture of the donor graft and temporary recipient immunosuppression with Cyclosporine A (CsA) on allograft survival. The adrenal medulla was transplanted immediately after dissection or after 1 week of culture, either at 24 degrees C or in the presence of 95% O2 at 37 degrees C. The results showed that neither cultural pretreatments affect the survival of the isografts as indicated by morphologic integrity of the grafts 30 days after transplantation. In the absence of in vitro culture of the donor medulla and short-term CsA recipient treatment, all the allografts were completely rejected at 30 days. Cultural pretreatment of the grafts either at 24 degrees C or in the presence of 95% O2, in conjunction with temporary CsA treatment of the recipient, or CsA treatment alone produced histologic survival of the grafts 30 days after transplantation. Varying degrees of lymphocytic infiltration were present in the grafts. When adrenal cortex was transplanted in conjunction with medullary tissue a bimodal immune reaction was observed; medullary tissue was infiltrated by lymphocytes, while the adrenal cortex remained morphologically intact with no infiltration at all. The experiments performed did not show a benefit in prolonging medulla allograft survival using pretransplant culture of the tissue.