Autotransplanted Animals Research Articles

Steroid-free maintenance of islet allografts using mycophenolate mofetil and cyclosporine in the non-human primate

Islet transplantation continues to be a promising treatment for type 1 diabetes, however, numerous limitations still prevent its widespread use. Many immunosuppressive medications used for islet transplantation are known to be diabetogenic. The goal of this study is to evaluate the short-term follow-up (90 day) of a steroid-free maintenance immunosuppression protocol of mycophenolate mofetil (MMF) and cyclosporine (CSA) in non-human primate islet allotransplantation. Diabetes was induced in the primate Macaca fascicularis via total pancreatectomy. Freshly isolated islets were autotransplanted (n=5) or allotransplanted (n=5) into the portal vein. Immunosuppression consisted of induction with rabbit anti-thymocyte globulin (ATG) and prednisone. CSA (25 mg/daily) and MMF (250 mg daily) were used for maintenance and given orally once daily throughout the 90 day study period. Fasting blood glucose measurements were used to monitor graft function. Intravenous glucose tolerance tests (IVGTT) were performed prior to pancreatectomy and at the study endpoint. Average fasting blood glucose levels were elevated in diabetic controls (313.6 mg/dl ±51.8) and untreated allograft recipients (257 mg/dl ±61.0) post-transplant. Auto-transplanted animals and allograft recipients treated with immunosuppression, on the other hand, maintained normoglycemia (74.5 mg/dl ±20.9, 62.3 mg/dl ±4.40) throughout the follow-up period. Additionally, beta cell function and first phase insulin secretion did not differ significantly between auto-graft and immunosuppressed allo-graft recipients post-transplant. We conclude that this steroid-free maintenance immunosuppression regimen is effective in maintaining islet allograft survival in the non-human primate and offers comparable graft function to that of autografts for up to 3 months post-transplant.

Comparison of the Portal Vein and Kidney Subcapsule as Sites for Primate Islet Autotransplantation

To date, the portal vein has been the primary site for clinical islet transplantation. Despite success, potential complications such as portal vein thrombosis still exist. The kidney subcapsule has been used successfully in rodent models of islet transplantation. We hypothesized that the kidney subcapsule as a site for islet transplantation in the nonhuman primate model would be as effective as the portal vein. Diabetes was induced in the primate Macaca fascicularis via a total pancreatectomy. Animals were kept under anesthesia during the isolation procedure. Islet isolation was performed using intraductal infusion with Liberase HI and mechanical digestion in the Ricordi chamber, and were purified using a continuous Ficoll gradient. Purified islets were autotransplanted either into the portal vein (n = 6) or the left kidney subcapsule (n = 5) of pancreatectomized animals. Intravenous glucose tolerance tests were performed prior to pancreatectomy and 10 days following transplantation. Three animals underwent pancreatectomy and served as diabetic controls. Of the six animals receiving islets in the portal vein, one developed portal vein thrombosis. All remaining autotransplanted animals in this group remained normoglycemic with glucose-induced insulin secretion that was not different from that prior to pancreatectomy. Of the five animals undergoing transplantation into the kidney subcapsule, only one maintained normoglycemia and elicited insulin secretion in response to glucose stimulation. The other four animals remained hyperglycemic. We conclude that the portal vein is superior to the kidney subcapsule as a site for islet transplantation in nonhuman primates 10 days posttransplantation.

Cellular and serological changes in the peripheral blood of splenectomized and spleen autotransplanted mice

Background Our department worked out a modified surgical form of spleen autotransplantation earlier, named “spleen apron method” introduced already into the clinical practice. Recently we tested the immunological changes in a group of patients autotransplanted with about 10–15% of their spleen, what was the at least always implantable amount after the severe splenic injuries. In the current work we aimed at measuring some cellular and serological changes in the peripheral blood of splenectomized and spleen autotransplanted inbred mice two and eight months after the operations in order to get more unambiguous results than that we could obtain in our patients with this technique. Materials and methods We divided 96 two months old Balb/c female mice into eight groups ( n = 12/group). The group of controls, sham operated, splenectomized and autotransplanted animals with two and eight months of survival time after the operations. During the autotransplantation we inserted the same amount of spleen, five slices, “chips,” about 10–15% of total mass of spleen, into the greater omentum similarly as it was used in the patients. The concentration of serum proteins were measured by laser nephelometry. The lymphocyte subsets were analyzed by flow cytometry. Results We found that two months after the operations the number of CD 19+ B-cells increased in the splenectomized but decreased in the autotransplanted animals. Eight months after the operations the number of both CD3+ T and CD19+ B lymphocytes decreased both in the splenectomized and autotransplanted animals compared to the controls and sham operated mice. However, the numbers of T and B cells were slightly but not significantly higher in the autotransplanted than in the splenectomized mice. The serum level of IgM was also decreased in the splenectomized and autotransplanted mice at both time points, however, eight months after the operations the concentration of IgM was significantly higher in the autotransplanted group than in the splenectomized animals. Conclusion The effects of autotransplanted “chips” were different at the various ages of the animals. Additionally, they showed some immunological benefit being quantitatively in accordance to the amount of the transplanted spleen. The elevated level of serum IgM what we found in the autotransplanted mice even with this amount of transplanted spleen eight months after the operations, however, might have the potentially greatest importance compared to splenectomy. These experiments can prove that the attempts for autotransplantation may have real perspectives but their efficacy depends on the amount of the successfully transplanted (saved) mass of spleen.

Distribution of peripheral blood cells in mice after splenectomy or autotransplantation

Our aim was to compare the distribution changes of peripheral leukocytes and erythrocytes in splenectomized and spleen-autotransplanted BALB/c female mice (n = 96), 2 and 8 months after surgery. In total, there were eight groups of animals: splenectomy, autotransplantation, sham, and untreated controls at both time points. We used the spleen-apron method of Furka et al. (Khirurgiia (Mosk) 1989;9:125-127), inserting five spleen chips into the greater omentum, for autotransplantation. Quantitative and qualitative blood cell counts and the phagocytic activity of cells (stimulated with zymosan) were determined. In splenectomized animals, the number of neutrophils significantly increased 8 months after surgery. The greatest phagocytic activity of neutrophils, however, was observed in autotransplanted animals of the same age. In splenectomized animals, erythrocyte volumes were significantly higher in the second postoperative month, but normalized by the eighth month. In conclusion, spleen autotransplantation has some beneficial effects, including clearing erythrocytes and preserving the phagocytic activity of neutrophils in peripheral blood.

Octreotide in the treatment of small intestinal dysfunction after a model of jejunoileal autotransplantation in the pig.

Enteric denervation and ischemic injury contribute to dysmotility and malabsorption following intestinal transplantation. We hypothesized that, by prolonging bowel transit and by ameliorating dysmotility, octreotide (OT) may improve cholesterol and bile acid absorption after jejunoileal autotransplantation. Seventeen pigs with fixed food intake underwent either jejunal transection (n = 6), or jejunoileal autotransplantation, which includes extrinsic autonomic denervation, lymphatic interruption, and in situ cold ischemia (n = 11). Five randomly chosen autotransplanted animals received intramuscular long-acting OT (10 mg) once a month. After 8 weeks, weight gain, intestinal transit time, fecal excretion of bile acids and cholesterol, and fractional cholesterol absorption were determined. Jejunal and ileal specimens were collected for histochemical analyses. Plasma cholestenol and campesterol, respective markers of cholesterol synthesis and absorption, were measured after 2 and 8 weeks. Following jejunoileal autotransplantation, octreotide treatment significantly increased the median intestinal transit time from 22.8 to 24.8 h and the median body weight gain from 166 to 187%. Jejunoileal autotransplantation significantly increased fecal bile acid excretion, plasma cholestenol, and bacterially modified fecal neutral sterols, and decreased absorption of cholesterol, plasma campesterol, and biliary cholesterol secretion. These changes were not significantly modified by OT treatment. Bowel wall and mucosal structure, mucosal proliferation, and weight or microvilli showed no statistically significant differences between autotransplanted animals with or without OT treatment. Findings of the present study suggest that octreotide prolongs intestinal transit time and improves weight gain after jejunoileal autotransplantation, but has no effect on malabsorption of cholesterol and bile acids.

Growth hormone selectively improves intestinal cholesterol absorption after jejunoileal autotransplantation in pigs

Background/Purpose Small bowel transplantation impairs enteric function and causes malabsorption of cholesterol and bile acids. Growth hormone stimulates intestinal absorptive function. The authors hypothesized that long-term growth hormone therapy could improve absorption of bile acids and cholesterol after autotransplantation of the jejunoileum. Methods Sixteen pigs with similar food, cholesterol, and fat intake underwent either sham laparotomy or a model of jejunoileal autotransplantation, including extrinsic autonomic denervation, lymphatic interruption, and in situ cold ischemia. Five randomly chosen autotransplanted animals received daily growth hormone treatment for 8 weeks. Serum lipids, absorption, and excretion of cholesterol, bile acids, and fat were determined after 8 weeks. Mucosal morphometrics, proliferation, and enzyme activities were determined. Plasma cholesterol precursors and plant sterols, respective markers of cholesterol synthesis and absorption, were measured after 2 and 8 weeks. Results After jejunoileal autotransplantation, growth hormone treatment significantly increased body weight gain, cholesterol absorption efficiency from 45.1% to 62.1%, plasma campesterol to cholesterol proportions, and biliary secretion of cholesterol. With or without growth hormone treatment, autotransplantation significantly increased fecal bile acid excretion, plasma cholesterol precursors, fecal bacterially modified neutral sterols, mucosal thickness of the ileum (but not jejunum), and intestinal transit time when compared with sham-operated animals. Crypt cell proliferation, mucosal enzyme activities, and microvilli showed no differences between the groups. Conclusions These findings suggest that growth hormone treatment selectively improves cholesterol, but not bile acid absorption, after autotransplantation of the jejunoileum.

The role of corticosterone in the metabolic recovery after intrasplenic adrenal autotransplantation in rats.

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.

ICAM-1 antisense oligodesoxynucleotides prevent reperfusion injury and enhance immediate graft function in renal transplantation

Ischemia-reperfusion injury after organ transplantation is a major cause of delayed graft function. We showed earlier that antisense oligodesoxynucleotides (ODN) for intercellular adhesion molecule-1 (ICAM-1) ameliorate reperfusion injury after acute ischemia. This study tested the hypothesis that antisense ODN for ICAM-1 prevents ischemia-reperfusion injury and facilitates immediate graft function in a rat autotransplantation model. Both kidneys were removed from male Lewis rats and re-implanted the left kidney after 30 minutes of cold ischemia time. The warm ischemia time was 60 minutes. Sham operated, uninephrectomized animals served as controls for renal function and histology. ICAM-1 antisense ODN (5 mg/kg), reverse ODN, or saline-vehicle were administered to donor animals i.v. six hours before autotransplantation. Glomerular filtration rate (insulin clearance), and serum creatinine concentrations were measured 24 hours post-transplantation. Tubular necrosis severity was assessed by histological grading scale. ICAM-1 expression was determined by immunohistochemistry and Western blot. Antisense ODN decreased ICAM-1 expression and leukocyte infiltration significant. Antisense ODN-treated animals showed significantly less tubular necrosis, than controls. Serum creatinine of antisense ODN-treated animals (N = 6) was 0.55 +/- 0.02 mg/dl compared to 1.92 +/- 0.07 mg/dl in reverse ODN-treated controls (N = 6; P < 0.01), 24 hours after transplantation. Antisense ODN-treated animals had normal GFR (0.93 +/- 0.07 ml/min/kidney wt) compared to sham-operated animals (0.95 +/- 0.09 ml/min/kidney wt), while autotransplanted animals treated with reverse ODN or saline-vehicle were all anuric. The ischemia-reperfusion-induced up-regulation of MHC class II was totally prevented by antisense ODN. ICAM-1 inhibition ameliorates ischemia-reperfusion injury and prevents delayed graft function. Antisense ODN-treatment of donors or donor organs for ICAM-1 may be useful for the prevention of reperfusion injury in human renal transplantation.

Modulation of autotransplanted adrenal gland by endothelin-1: a morphological and biochemical study.

Adrenal gland autotransplantation, a model of cortical tissue regeneration, provides the reconstruction of distinct functional and morphological zonae. A morphological and biochemical study of the adrenal gland of adult male rats after autotransplantation and endothelin-1 (ET-1) administration was made. The technique involved bilateral adrenalectomy and placement of pieces of the adrenal gland in a dorsal plane between the skin and muscle. The animals were killed 90 days after the autotransplantation and 1 hr after intravenous ET-1 administration (0.5 microgram/kg body weight). The autotransplanted pieces were removed, fixed, and processed for light and electron microscopic morphologic studies. Trunk blood was collected for steroid assay. Saline-treated control autotransplanted animals showed no remarkable differences in adrenal organization; grafts exhibiting a mass of regenerated cortical tissue were arranged in nests of glandular cells surrounded by a fibrous capsule and intersected by layers of connective tissue. The adrenal medulla was systematically absent. Ultrastructure of ET-1-treated animals revealed an inner area in the graft, consisting mainly of fasciculatalike cells. Cytoplasmic changes were evident, with high variations in mitochondrial size and arrangement. Profiles of smooth endoplasmic reticulum sometimes exhibited evidence of hypertrophy. Glandular cells in the graft outer area (subcapsular) were almost invariably like glomerulosa; however, some of them showed mitochondria with a peculiar arrangement of the cristae. "Hybrid" cells with mitochondria resembling those of the zona reticularis were also observed in the subcapsular environment. ET-1-stimulated animals showed significant increases in plasma corticosterone and aldosterone concentrations. Endothelin-1, previously reported to stimulate acutely the aldosterone secretion by the adrenal zona glomerulosa in the rat, seems to exert a modulator role on the physiology of adrenal autotransplants, their regeneration and secretion.

New insights into zonal differentiation of adrenal autotransplants in the rat: an immunohistochemical study

Adrenal gland autotransplantation, an interesting model of adrenal regeneration, provides the reconstruction of distinct functional and morphological zonae. An immunohistochemical study of the adrenal gland of adult male rats after autotransplantation and endothelin-1 (ET-1) stimulation was carried out. The technique involved total adrenalectomy and immediate autotransplantation of small adrenal pieces under the skin of the dorsal region. The animals were killed 90 days after the autotransplantation and 1 h after intravenous ET-1 administration. Sections of recovered adrenal grafts were incubated with IZAb, a monoclonal antibody which interacts with an antigen (IZAg) predominantly found in rat adrenal inner zones. Saline-treated control autotransplanted animals showed IZAb immunostaining in almost all adrenocortical tissue, with the exception of small clusters of cells beneath the capsule. ET-1-treated animals exhibited an extended zone devoid of immunostaining and located in the subcapsular area. In addition, ET-1-stimulated animals showed significant increases in aldosterone as well as corticosterone concentrations in plasma. These results revealed that ET-1 stimulated the development of an extended subcapsular zone lacking IZAg expression, an effect that suggests its role in zona glomerulosa induction in these animals.

Autotransplantation of the adrenal cortex: a morphological and autoradiographic study.

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.

Autotransplantation of adrenal cortex: An autoradiographic study

In previous experiments we demonstrated that 4, 12 and 24 weeks after rat adrenal cortex autotransplantation, the animals survived and the autotransplant was easily reproducible. It was observed that 4 weeks after transplantation the plasma corticosterone concentration (0.20 μg/ml) was already comparable to that in controls and that the light and electron microscopy features of the cells of the glomerulosa and fasciculata zones were similar to those of adrenal cells in control animals.In order to clarify the origin of glandular cells in the autotransplanted animals, autoradiographic experiments were made. Four male rats with body weights ranging from 180to195 gm underwent bilateral adrenalectomy via two dorsal incisions made along the lateral border of the dorsal muscle mass. Pieces of adrenals were autotransplanted immediately under the skin of the dorsal region. Four animals were “sham-operated” without removal of adrenals and served as controls. The animals were fed “ad libitum” and with saline solution, and were sacrificed at 2,3,7 and 15 days. 3H-Thymidine (1 μCi/gm body weight) was administrated intraperitoneally to each animal two hours before the sacrifice. Necropsy was carried out in all animals in order to search for accessory adrenals. The autotransplanted pieces were removed, fixed and processed for LM and EM morphologic and autoradiographic studies. For LM autoradiographic studies Ilford K5 nuclear emulsion was used, and the sections exposed for 33 days; for EM autoradiographic studies Ilford L4 nuclear emulsion was used, the sections exposed for 90 days, and the radio autographs developed with Kodak D 19b.

Pituitary-Adrenal Stress Response in the Absence of Brain-Pituitary Connections

To examine whether an acute pituitary-adrenal response to stress may occur in vivo in the absence of hypothalamic-pituitary connections, we measured plasma beta-endorphin (beta-EP) and corticosterone (C) in rats after acute thermal injury. beta-EP rose significantly after thermal injury in normal rats and rats bearing pituitary-to-kidney autotransplants but not in animals with pituitary aspiration without reimplantation. Corticosterone responses paralleled beta-EP but were significant only in normal controls. Propranolol pretreatment did not reduce postburn beta-EP and C rises in autotransplanted animals. Therefore, since circulating factors contribute in vivo to pituitary-adrenal responses, the widespread practice of using "stress hormone" responses to quantitate perioperative stress or pain may in some circumstances be flawed.

Possible Direct Effect of Serotonin on Pituitary Prolactin Secretion: in vivo and in vitro Studies

In this work we analyze the possibility of serotonin (5-HT)-releasing prolactin (PRL) through a direct action at the pituitary level. 5-HT (2 mg/kg i.v.) stimulates PRL secretion in hypophysectomized autotransplanted animals (HAG) significantly and this effect was not influenced by pretreatment with the dopaminergic antagonist domperidone. In perifused pituitaries, 5-HT administration (0.01, 0.1 and 1 microM for 90 min, or 1, 10, 100 microM for 15 min) was ineffective in stimulating PRL release. In pituitaries obtained from animals previously treated with the neurotoxic 5,7-dihydroxytryptamine (5,7-DHT) or vehicle and incubated in the presence of 5-HT (2.5, 5 and 10 microM), no response in PRL secretion was observed. These results suggested that 5-HT does not release PRL through a direct pituitary action, and that the effect observed in HAG animals could be mediated through the release of a PRL-releasing factor after 5-HT administration.

Intermediary metabolism in diabetic dogs treated with pancreatic autotransplants and insulin pumps

Diurnal metabolite profiles were studied in pancreatectomized dogs who had received grafts of their own pancreas. The results were compared to similarly diabetic animals who received exogenous insulin pumped intravenously either peripherally or portally. All animals were studied at least 52 weeks after pancreatectomy. Nondiabetic animals were similarly studied as normal controls. Interestingly, all of the diabetic animals were in excellent metabolic control but significant differences from normal in one or more of the metabolic variables existed in each group. Thus, none of the methods of insulin replacement entirely restored to normal the metabolic state of the pancreatectomized dogs. For example, in the autotransplanted animals, the profiles of glucose, lactate, pyruvate, and alanine manifested either abnormal levels and/or abnormal postprandial excursions. The grafts delivered insulin into the peripheral rather than portal circulations. In the peripherally infused animals, the profiles of pyruvate, alanine, and free fatty acids were also somewhat abnormal. In the portally infused animals the profiles only of pyruvate and alanine showed minor abnormalities without clear statistical significance in some respects as well. The other differences observed were statistically significant ( P < 0.05–0.01). These results suggest that the portal route of insulin delivery may be the route of choice if intermediary metabolism is to approximate normal most closely whether exogenous intravenous insulin is replaced by implanted pumps or endogenous insulin is replaced by pancreatic transplants and that none of these futuristic methods of insulin replacement entirely restore to normal the metabolic state of pancreatectomized dogs.

Metabolic Control in Diabetic Dogs Treated with Pancreatic Autotransplants and Insulin Pumps

Fasting metabolite and hormonal levels were studied prospectively in pancreatectomized dogs who had received grafts of their own pancreas. The results were compared with similarly diabetic animals who received exogenous insulin pumped intravenously either peripherally or portally. All animals were studied for 48-91 wk after pancreatectomy. In the autotransplanted animals, the fasting levels of glucose, lactate, pyruvate, alanine, pancreatic glucagon, insulin, gastric inhibitory peptide, and pancreatic polypeptide were all abnormal. In the peripherally infused animals, the fasting levels of glucose, pyruvate, alanine, free fatty acids, and insulin were also abnormal. In the portally infused animals, pyruvate, alanine, gastric inhibitory peptide, gastrin, and pancreatic polypeptide were abnormal. These results suggest that the portal route of insulin delivery may be necessary if fasting metabolite and hormonal levels are to approximate normal most closely whether exogenous intravenous insulin is replaced by implanted pumps or endogenous insulin is replaced by pancreatic transplants.

The morphology of the mucous gland and its responses to prolactin in the skin of the red-spotted newt.

The mucous gland of the red-spotted newt, Notophthalamus viridescens viridescens, Rafinesque was examined by histochemical and ultrastructural techniques and its cytological responses to various hormonal conditions were studied. Its secretory epithelial cells produce and release in merocrine fashion a neutral, unsulphated mucosubstance. The secretory epithelium is bounded peripherally by a thin, but apparent non-functional, myoepithelium. The duct of this mucous gland consists of a single keratinized tubular cell that extends from the neck region of the gland to the surface of the epidermis. Mucous secretion is absent or greatly reduced on the skins of newts maintained under laboratory conditions for a few weeks but reappears after injection of ovine prolactin. Mucous glands in laboratory conditioned animals show a 4-fold increase in volume brought about by the engorgement of their epithelial cells with secretory granules. Ovine prolactin reduces the volume of the glands to unconditioned levels with a corresponding reduction in granular content, suggesting that prolactin functions in the release of the granules. This view is reinforced by the findings that autotransplantation of the pituitary gland prevents the conditioning effect and that glandular volume increases in autotransplanted animals given ergocornine. Granular accumulation begins also in hypophysectomized newts but ceases after a week, indicating the need for some hypophyseal factor in the synthesis as well as the release of the granules. Ovine prolactin restores mucous glands of hypophysectomized newts to the unconditioned state. Contrary to earlier findings, ovine prolactin induces a reduction in the volume of the mucous gland in thyroidectomized newts.