Growth of forelimb allografts in young rabbits immunosuppressed with cyclosporine

Seventeen forelimbs were transplanted orthotopically from young Dutch rabbits to young New Zealand rabbits treated with cyclosporine. The transplanted limbs demonstrated significant bone growth. The growth in the transplanted limbs was about 75 to 80% of that observed in the unoperated limb. The long bones of the 3 longest surviving rabbits (133 days, 150 days, 150 days) studied radiographically demonstrated increases in length over their original lengths (humerus 22%, ulna 26%, and radius 31%). Hair and nail growth were noted at about day 10. Response to pain stimuli (withdrawal of forelimb) and functional use (ambulation with 50% weight bearing) was seen at two to three months. Permanent survival was not achieved because of a species-specific toxic wasting syndrome from cyclosporine.     

Effects of immunosuppression induced by thalidomide and cyclosporine in heterotopic heart transplantation in rabbits

Because of its anti-inflammatory and immunodepressive effects, thalidomide has been used for the treatment of dermatologic diseases and of host-versus-graft reactions in patients undergoing bone marrow transplantation. We evaluated the immunosuppressive action of thalidomide alone or in combination with cyclosporine on the prevention of rejection of heterotopic cardiac allografts in rabbits. METHODS: Fifty rabbits were used including 25 donors and 25 recipients. Recipient animals were divided into five groups (n = 5 each): group 1 (control), non-immunosuppressed animals; group II, animals immunosuppressed with cyclosporine (10 mg/kg per day); group III, immunosuppressed with thalidomide (100 mg/kg per day); group IV, immunosuppressed with cyclosporine (5.0 mg/kg per day); and group V, immunosuppressed with cyclosporine (5.0 mg/kg per day) in combination with thalidomide (50 mg/kg per day). The medications were administered through an orogastric catheter starting on the day before the transplant. The heart of the donor was implanted into the recipient's abdomen. RESULTS: The combination of thalidomide and cyclosporine showed the lowest histopathological rejection score (P < .05). Thalidomide administered alone or in combination with cyclosporine was effective against rejection, significantly increasing survival (P < .01). CONCLUSIONS: Thalidomide may be considered to be an adjuvant immunosuppressant.     

Effects of thalidomide, cyclosporine, and diclofenac on skin allograft survival in rabbits

The present study evaluated the effects of thalidomide, cyclosporine, and diclofenac on skin allograft survival in 42 rabbits divided into the following groups (n = 6): group 1, autograft control; group 2, allograft control; group 3, allografts under thalidomide (100 mg/kg/d); group 4, allografts under sodium diclofenac (2 mg/kg/d); group 5, allografts under cyclosporine (10 mg/kg/d); group 6, allografts under cyclosporine (5 mg/kg/d); group 7, allografts under cyclosporine (5 mg/kg/d) plus thalidomide (100 mg/kg/d). The drugs were given via the orogastric tube the day before transplantation and daily during the postoperative period. Total circular skin grafts from the ear were exchanged between California and White New Zealand rabbits. Cyclosporine (10 mg/kg/d) increased allograft survival, an effect that was comparable to cyclosporine (5 mg/kg/d) plus thalidomide (100 mg/kg/d). Thalidomide and diclofenac given alone had minimally significant effects on the mean survival of skin allografts. The number of eosinophils around the necrotic skin was higher in the diclofenac group. The group receiving cyclosporine combined with thalidomide displayed the lowest number of eosinophils surrounding the allograft. In conclusion, the combination of thalidomide and cyclosporine in subtherapeutic doses may be useful for the treatment of skin allografts.     

Transplantation of epiphyseal plate allografts between animals of different ages.

The purpose of the experiment was to study growth of epiphyseal plate allografts after transplantation into subjects of a different age, thus preparing for future transplantation of epiphyseal plate or extremity allografts in children. Microvascular transplantation of proximal tibial epiphyseal plate allografts was performed in skeletally immature New Zealand White female rabbits. The growth of 9-week-old epiphyseal plate allografts was examined in both 9-week-old and 17-week-old recipients, as was the growth of 17-week-old epiphyseal plate allografts in 17-week-old recipients. Immunosuppression was with cyclosporine (Cyclosporine A). Successful transplants were confirmed with 99mTc-MDP isotope scanning, and growth was evaluated with weekly standardized radiographs until death. Growth rate was found to depend on the age of the donor epiphyseal plate and was independent of the age of the recipient. This has clinical implications for the procurement of donor tissue in potential transplantation of epiphyseal plate allografts in children.     

A study of the mechanisms influencing ligament growth

This study investigated the effects of tension and/or exogenous growth hormone on the growth (elongation) of ligaments. In the first experiment, tension was applied to the lateral collateral ligaments of immature rabbits. In the second experiment, a group of skeletally mature rabbits was given exogenous growth hormone while tension was applied to their lateral collateral ligaments. The results revealed that immature rabbit ligaments elongated 140 +/- 18% when tension was applied, while "control" ligaments elongated 79 +/- 5% (P less than .01). In mature rabbits receiving exogenous growth hormone, no significant change was found in the ligament's length with or without the application of tension when compared with controls. Tension applied to ligaments in immature rabbits can increase ligament growth, indicating that physical forces (tension) may be important in the regulation of ligament growth. The same tension applied to mature rabbit ligaments in combination with exogenous growth hormone did not cause a resumption of growth, indicating that tension and the presence of growth hormone are not the only factors necessary for ligament growth.     

Adriamycin cytotoxicity may stimulate growth of hepatocellular tumours in an experimental model for adjuvant systemic chemotherapy in liver transplantation

Adjuvant treatment with adriamycin has been suggested to improve results after liver transplantation for hepatocellular cancer. Here we have applied an animal model for evaluation of treatment with adriamycin and/or cyclosporine A on liver tumour growth. Three chemically induced rat liver tumours with various degree of differentiation were transferred to the spleens of syngenic rats. Each recipient group was divided into four subgroups, treated with adriamycin and/or cyclosporine A or none of the drugs. When the tumour was well differentiated no proliferation was found in any of the subgroups. When the tumour exhibited a more pronounced dysplasia, adriamycin stimulated tumour growth. This effect was further increased by cyclosporine. In the animals transplanted with the most aggressive tumour, adriamycin inhibited tumour growth. When given together with cyclosporine this inhibition was counteracted. These data suggest that adriamycin, especially when given together with cyclosporine, may have a stimulatory effect on liver tumour cell growth.     

Immunomodulators interfere with angiopathy but not vasospasm after subarachnoid haemorrhage in rabbits

Summary The present study deals with the effects of immunomodulators on the morphology of intracerebral arterial walls in rabbits with experimental subarachnoid haemorrhage (SAH). Immunostimulators: thymostimuline and inosine dimethylamino-isopropanol-p-acetamido-benzoate were found to aggravate the angiopathic changes, whereas immunosuppressive drugs — cyclosporine A and azathioprine appeared to prevent the damage. The authors consider the possibility of using immunosuppressive drugs in patients with ruptured intracranial aneurysms.     

In vitro cyclosporine toxicity. The effect of verapamil

The epithelial cell line LLC-PK1, which expresses many proximal tubular characteristics, was used to investigate the relationship between calcium, the calcium channel blocker verapamil, and cyclosporine toxicity. The LLC-PK1 cells took up cyclosporine when this was added in a concentration of 2 micrograms/ml, and this uptake was maximal at 30 min (112 +/- 3 ng cyclosporine/mg cell protein). At 12 micrograms/ml it inhibited the sodium glucose cotransporter, as assessed by phlorizin-inhibitable 14C-alpha-methyl glucopyranoside (alpha-MG) uptake (control 37.2 +/- 6.3, 12 micrograms/ml 21.2 +/- 1.1 mumol/hr/mg protein). Cyclosporine at 2 micrograms/ml did not affect cell growth after 5 days (control 945 +/- 60 micrograms cell protein per 25 cm2 flask, 2 micrograms/ml cyclosporine/ml 1046 +/- 32 micrograms protein/flask), even in the presence of 7.6 mM ionized calcium (862 +/- 37 micrograms protein/flask). Cyclosporine at 12 micrograms/ml inhibited cell growth (286 +/- 27 micrograms protein/flask), and raising the ambient ionized calcium concentration to 7.6 mM reduced cell growth further (91 +/- 6 micrograms protein/flask). Cyclosporine at concentrations of 2 and 12 micrograms/ml produced increasing cell vacuolation, as seen in vivo. Short-term uptake of 2 micrograms/ml cyclosporine could be inhibited by 1.0 mM and 0.5 mM verapamil (49 +/- 9.5 and 71 +/- 6.4 ng cyclosporine/mg cell protein, respectively, at 30 min). However, in the presence of 2 micrograms/ml cyclosporine 0.1 mM verapamil was toxic to the cells grown over five days (44 +/- 5 micrograms protein/flask). At 0.01 mM verapamil was not toxic to cell growth (921 +/- 29 micrograms protein/flask), but raising the medium calcium to 7.6 mM reduced cell growth (652 +/- 96 micrograms/ml). Inhibition of cyclosporine uptake did not occur with 0.01 mm verapamil (control 145.6 +/- 12.3 vs. 0.01 mM verapamil 150.4 +/- 3.8 ng cyclosporine/mg cell protein). The LLC-PK1 cell line represents a good in vitro model for cyclosporine renal tubular toxicity, as the in vivo observation of glycosuria and proximal tubular cell vacuolation in cyclosporine nephrotoxicity can be reproduced. In vitro this is shown to be associated with inhibition of sodium-dependent glucose cotransport. Verapamil inhibited cyclosporine uptake, but only at concentrations that were toxic to the cells. Verapamil potentiated rather than reduced the increased cyclosporine toxicity produced by increasing the medium calcium concentration. The suggested protective effect of verapamil against cyclosporine nephrotoxicity is therefore unlikely to be due to inhibition of cyclosporine uptake or of calcium entry into proximal tubular cells.     

Differential effects of modern immunosuppressive agents on the development of intimal hyperplasia

Modern immunosuppressive agents such as tacrolimus and rapamycin are claimed to be associated with a reduction in vascular narrowing, a central feature of chronic rejection. This study assesses the effect of cyclosporine, tacrolimus and rapamycin on the development of intimal thickening, fibrosis-associated genes and deposition of extracellular matrix (ECM) proteins in a model of intimal hyperplasia. Male Sprague-Dawley rats received either no treatment or 5 mg/kg cyclosporine, 0.1 mg/kg tacrolimus or 0.05 mg/kg rapamycin. Animals underwent left common carotid balloon angioplasty, and intima medial ratios, pro-fibrotic gene expression and ECM accumulation were calculated at 14 and 28 days. Cyclosporine was associated with increased intimal thickening compared to controls (P<0.004). Tacrolimus had no effect on intimal thickening, whilst rapamycin significantly inhibited intimal thickening at both 14 and 28 days (P<0.004 and P<0.026, respectively). All groups significantly inhibited matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1, transforming growth factor (TGF)- and collagen III expression at 14 days (P<0.001), but increased ECM deposition. However, rapamycin marginally reduced ECM deposition compared to cyclosporine (P<0.06). Treatment with cyclosporine was associated with worsening of vascular narrowing, whilst rapamycin showed a beneficial reduction in intimal thickening. Treatment with all immunosuppressive agents resulted in increased ECM deposition. Rapamycin may halt the progression of vascular narrowing compared to both cyclosporine and tacrolimus.     

Beneficial effect of cyclosporine on renal transplantation. A multicenter long-term study

Data were collected prospectively on 8581 cadaveric renal transplants performed by institutions of the South-Eastern Organ Procurement Foundation (SEOPF) during the period November 1, 1983 through December 31, 1987. Cyclosporine was the initial then always used immunosuppressant for 5742 of these patients while 1050 never received cyclosporine. The drug was started late in the course of 481 transplants and stopped early in 378 cases. This allowed for 7651 transplants to be analyzed regarding these four categories of cyclosporine use or non-use. Actuarial graft survival for the cyclosporine "ALWAYS" group was 75% at one year, 68% at two years, 62% at three years, and 59% at four years compared with 55%, 49%, 45%, and 43%, respectively for the cyclosporine "NEVER" group (P less than 0.0001). Inclusion of the 930 cases that could not be categorized regarding cyclosporine use or for which actuarial data was not complete allowed all 8581 transplants to be analyzed by multivariate methods. This analysis disclosed significant effects on graft survival due to delayed graft function, prior transplant, recipient race, HLA match, level of PRA, and cyclosporine use. Organ sharing had no effect on graft outcome. While cyclosporine improves outcome in renal transplantation, the importance of other biologic factors affecting graft survival is not diminished by its use.     

Myocyte hypertrophy in the transplanted heart. A morphometric analysis

In order to better define long-term changes in the transplanted heart with respect to the effects of cyclosporine and the ischemic time of the donor heart, endomyocardial biopsies were examined ultrastructurally from 20 cardiac transplant recipients three years posttransplantation. The biopsies were divided into four groups of five based on the donor heart ischemic time in "on-site" versus "distantly procured" hearts and on the immunosuppression protocol: group A: "on site" donor hearts and cyclosporine-based immunosuppression; group B: "on site" donor hearts with conventional immunosuppression (azathioprine-based immunosuppression without cyclosporine); group C: distantly procured donor hearts treated with cyclosporine; and group D: distantly procured donor hearts treated with conventional immunosuppression. All four groups showed a significant increase in the average width of myocytes when compared with normal myocardium, (group A, P less than 0.05; groups B, C, D, P less than 0.01). Also, there was a significant difference between the average widths of myocytes from on-site donor hearts and distantly procured donor hearts (P less than 0.04). There was no significant difference between the average myocyte widths of groups treated with cyclosporine and those with conventional immunosuppression. This study shows that despite the hypertension induced by cyclosporine, myocyte hypertrophy at 3 years posttransplantation does not appear to be significantly greater than in patients treated with conventional immunosuppression. Distantly procured donor hearts have more hypertrophy. Due to the increasing evidence that cardiac hypertrophy per se may predispose to serious ventricular arrhythmias, this study supports the use of on-site as opposed to distantly procured donor hearts.     

Retardation of renal growth and ornithine decarboxylase activity by cyclosporine after uninephrectomy in rats

Renal transplantation or a partial reduction in renal mass prompts compensatory growth of the kidney, an event that appears to be stimulated by a circulating substance, the purported renotropic factor. This factor may stimulate synthesis of renal polyamines by induction of the rate-limiting enzyme in polyamine metabolism, ornithine decarboxylase. Cyclosporine has been shown to attenuate induction of renal ornithine decarboxylase activity in response to other tropic hormones, such as prolactin, thyroxine, and dexamethasone. These studies were undertaken to evaluate the effects of cyclosporine on induction of renal ornithine decarboxylase activity and growth in response to the serum renotropic factor. Rats were given either 15.0 or 25.0 mg/kg/day or cyclosporine for six days following removal of one kidney. The growth of the remaining kidney was reduced by cyclosporine treatment compared with pair-fed, vehicle-treated control animals. Administration of 25.0 mg/kg/day of cyclosporine also reduced the activity of ornithine decarboxylase in the growing kidney. We conclude that cyclosporine imposes a limitation on the ability of the kidney to grow in response to a reduction in renal mass in rats. The mechanisms of this effect may relate to a blunting of the induction of renal ornithine decarboxylase activity in response to the renotropic factor.     

The efficacy of adjuvant cytostatic therapy after organ transplantation for malignancy: an experimental study with a combined transplantation/tumor model

New data show that perioperative cytostatic therapy is beneficial in the case of liver transplantation for hepatic cancer. However, it has not been established clearly whether chemotherapy interferes with graft rejection. We therefore studied the interactions between tumor growth and graft rejection, especially with regard to chemotherapy, using a combined tumor/transplantation model. As a tumor model, we used the Novikoff hepatoma, a malignant hepatoma that was injected subcutaneously into the backs of rats. Heterotopic heart grafting served as the transplantation model. In a first step (a), we studied the effect of cytostatic therapy on tumor growth: tumor cells were injected, and in four groups epirubicin, cyclosporine, epirubicin + cyclosporine, and placebo were applied, in corresponding groups, transplantation was additionally performed. Tumor growth was measured and the resected tumors were examined by histology and immunohistology. In a second step (b), we studied the effect of chemotherapy on graft rejection: transplantation was performed and the above-mentioned drugs were applied; in corresponding groups, a solid tumor was additionally induced and resected immediately before transplantation. The results of these procedures were as follows: (a) Epirubicin decreased tumor growth and diminished the volume-increasing effect of cyclosporine significantly. After transplantation, tumor growth was similar. (b) Epirubicin prolonged graft survival significantly, and the combination with cyclosporine had an augmenting effect. In the corresponding groups, graft survival was similar. In conclusions. chemotherapy diminishes the tumor-increasing effect of cyclosporine and does not interfere negatively with graft survival. It might therefore be beneficial after transplantation for malignancy. Received: 14 December 1998 Revised: 23 August 1999 Accepted: 8 November 1999     

Adult respiratory distress syndrome and convulsions associated with administration of cyclosporine in liver transplant recipients

A "capillary leak" syndrome resulting from cyclosporine-induced membrane toxicity has been postulated as the cause of convulsions and pulmonary edema in bone marrow transplant recipients. We describe here the occurrence of similar complications in a group of 21 adults receiving liver transplants since July 1982. Of 12 patients treated with i.v. cyclosporine (4 mg/kg/day), 2 developed an adult respiratory distress syndrome (ARDS) within five days of the operation, but it was not found in those given prednisolone (0.05-1.0 mg/kg/day) and azathioprine (1.0 mg/kg/day). ARDS only occurred when cyclosporine was administered through a central vein, and therefore might be related to high concentrations of cyclosporine reaching the pulmonary circulation and causing damage to vascular membranes. Convulsions occurred in one patient given i.v. cyclosporine, and in three when therapy was changed and cyclosporine and corticosteroids were used in combination. Convulsions did not occur inthe same patients as ARDS, were not part of a generalized "capillary leak" syndrome, and were not associated with hypertension or renal failure, as reported elsewhere in children. Fluid retention consequent on cyclosporine administration aggravated by the use of corticosteroids appears to be the most likely explanation of the convulsions.     

Effects of Cyclosporine on the Antioxidant Status and Oxidative Stress in the Glioma Cells

Objectives

After organ transplantation, some patients suffer neurological complications. Among the immunosuppressants, cyclosporine can cause neurological side effects. However, the mechanisms of encephalopathy by cyclosporine are not fully understood. We measured the antioxidant status, the hydrogen peroxide level, and the malondialdehyde level in glioma cells after cyclosporine treatment.

Methods

The production of hydrogen peroxide was determined using a modified xylenol orange method. The amount of malondialdehyde was measured using the thiobarbituric acid assay. Total antioxidant status was measured using Free Radical Analytical System 4 with kits.

Results

Cyclosporine resulted in the production of hydrogen peroxide by the glioma cells. The increased production of hydrogen peroxide depended on the drug concentration. The antioxidant status was decreased in the glioma cells after cyclosporine treatment. The malondialdehyde level was not changed in glioma cells after cyclosporine treatment.

Conclusions

Increased production of reactive oxygen species and decreased antioxdant status by cyclosporine in glioma cells may contribute to neurological side effects in transplantation patients.     

Combined nephrotoxic effects of cyclosporine and endotoxin

This study presents experimental evidence that cyclosporine (CsA) potentiates the nephrotoxicity of endotoxin. This study was motivated by clinical observations in 4 cyclosporine (CsA)-treated renal allograft recipients who developed severe, and sometimes irreversible, nephrotoxicity after infections. CsA or vehicle was administered intramuscularly to rabbits for 5 days, and subsequently both groups of animals received one dose of endotoxin intravenously. Compared with controls, CsA-treated animals demonstrated significantly higher elevations of blood urea nitrogen and serum creatinine 24 hr after endotoxin. By contrast, both groups of animals developed similar degrees of thrombocytopenia. Histologic evaluation of kidney tissues 24 hr after endotoxin revealed significantly greater tubular toxicity and a higher glomerular polymorphonuclear leukocyte (PMN) infiltration in CsA-treated animals. Semiquantitative scores of tubular damage correlated directly with the mean number of PMN/glomeruli in both groups of animals. Immunofluorescent microscopy of kidney tissues was negative for fibrinogen and for complement deposition in both CsA and control groups. We conclude that CsA enhances endotoxin nephrotoxicity in rabbits. This effect does not appear to be mediated by activation of coagulation factors. However, a role for PMN is suggested. CsA should be used with caution in patients with deteriorating renal function who are suspected of having severe bacterial infections.     

Pharmacokinetically determined cyclosporine dosage in young children

To account for the individual variability in cyclosporine pharmacokinetics and the non-existence of dosing recommendations in young children, we studied the pharmacokinetics of cyclosporine before renal transplantation in ten children aged 1.1–2.5 years, to determine the appropriate individual dose. Our aim was to reach a steady-state cyclosporine blood level of 200–300 g/l, 8 h after a dose in the first days after renal transplantation. Cyclosporine was given as a single oral dose (10 mg/kg) or as a 4-h i. v. infusion (3 mg/kg), and the blood concentration was determined for 24 h by a specific monoclonal radioimmunoassay. The mean terminal cyclosporine half-life (t _1/2) was 9.3 h (range 2.8–20.4), blood clearance 10.8 ml/min per kilogram (range 6.8–22.7) and volume of distribution 2.8 l/kg (range 1.4–4.7). The bioavailability of oral cyclosporine was low; the mean amount absorbed was 21.8% of the administered dose (range 11–35). The mean calculated dose needed to attain the intended predose blood cyclosporine level of 200–300 g/l at steady-state was 5 mg/kg per day for i.v. and 21 mg/kg per day for oral administration. In view of the shortt _1/2, we used three doses/day. The validity of the predicted doses is shown by the mean cyclosporine doses used during the first 10 days after transplantation, which were 93.5% of the calculated oral and 96.6% of the calculated i.v. doses. The observed mean cyclosporine concentration during the same period was 196 g/l.     

Alpha adrenergic antagonism by cyclosporine.

Cyclosporine has been shown to exert antiadrenergic actions in previous studies from this laboratory. Interactions of CsA with the alpha-one and alpha-two receptor were studied in rabbits. Blood pressure studies revealed that CsA exerts a competitive, reversible antagonism against norepinephrine, epinephrine, and phenylephrine. Further studies of heart rate depression by clonidine also demonstrated that CsA antagonizes this action. In conclusion, cyclosporine appears to act as a nonspecific alpha antagonist.     

Dorsal arthrodesis in prepubertal New Zealand white rabbits followed to skeletal maturity: Effect on thoracic dimensions, spine growth and neural elements

Background:

Several studies have shown that severe spinal deformity and early arthrodesis can adversely affect the development of the spine and thorax by changing their shape and reducing their normal function. This article analyzes the consequences of posterior fusion on the growth of spine, thorax and neural elements in New Zealand white rabbits and compares with similar human data.

Materials and Methods:

The first section of the article analyzes the consequences of T1-T6 dorsal arthrodesis on the growth of the spine, sternum, thorax volume and neural elements in 12 prepubertal female New Zealand white rabbits, through a study of CT scans and histology specimens. The second part, evaluates thoracic dimensions in 21 children with spinal arthrodesis for treatment of deformity performed prior to nine years of age.

Results:

Dorsal arthrodesis in prepubertal rabbits changes thoracic growth patterns. In operated rabbits thoracic depth grows more slowly than thoracic width. The sternum as well as length of thoracic vertebral bodies in the spinal segment T1-T6 show reduced growth. Children undergoing spinal arthrodesis before nine years of age were noted to have shortened height, short trunk and disproportionate body habitus at skeletal maturity. Observed spine height and chest dimension values were reduced compared to the expected norms. The ratio between chest width and chest depth was below normal values.

Conclusions:

The first part of the study shows that thoracic dorsal arthrodesis in prepubertal New Zealand white rabbit influences thoracic, spine growth and affects the shape of pseudo unipolar neurons of the dorsal root ganglia. The second part demonstrates that children treated before nine years of age have significantly reduced spine height and thoracic dimensions. The thorax becomes elliptical as chest depth grows less than chest width. Both experimental and clinical findings contribute to explain reduced chest growth and subsequent thoracic growth disturbance in patients treated with early arthrodesis.