Wistar-Furth Rats Research Articles

Hepatic cholangiodysplastic pseudocirrhosis with multiple disseminated intrahepatic nodules of pancreatic tissue

We developed an inbred rat model of diabetic embryopathy, in which the offspring displays skeletal malformations (agnathia or micrognathia) when the mother is diabetic, and no malformations when she is not diabetic. Our aim was to find genes controlling the embryonic maldevelopment in a diabetic environment.We contrasted the fetal outcome in inbred Sprague-Dawley L rats (20% skeletal malformations in diabetic pregnancy) with that of inbred Wistar Furth rats (denoted W, no skeletal malformations in diabetic pregnancy). We used offspring from the backcross F1 × L to probe for the genetic basis for malformation of the mandible in diabetic pregnancy. A set of 186 fetuses (93 affected, 93 unaffected) was subjected to a whole genome scan with 160 micro satellites. Analysis of genotype distribution indicated 7 loci on chromosome 4, 10 (3 loci), 14, 18, and 19 in the teratogenic process (and 14 other loci on 12 chromosomes with less strong association to the malformations), several of which contained genes implicated in other experimental studies of diabetic embryopathy. These candidate genes will be scrutinized in further experimentation.We conclude that the genetic involvement in rodent diabetic embryopathy is polygenic and predisposing for congenital malformations.

Cytotoxic T-cell elimination during anti-CD4-induced rat liver acceptance and rapid replacement of functional graft antigen-presenting cells.

In previous studies, we showed that primed T cells were eliminated in long-term survival Wistar Furth (WF) recipient rats with spontaneously accepted Lewis (LEW) liver graft and that the grafted liver lost the ability to elicit rejection reaction early after liver transplantation. We hypothesized that the same phenomenon may be observed in tolerant animals after immunosuppression in a rejector rat strain combination (WF-->LEW). Furthermore, we proposed the repopulation of liver allograft with host antigen-presenting cells rapidly after transplantation. Recipient LEW rats that underwent anti-CD4 therapy accepted the WF liver allografts after a transient rejection reaction. In tolerant animals, alloreactive CD8 T cell precursors were present, but primed T cells were absent. Intraperitoneal challenge with grafted WF liver homogenates obtained from recipient LEW rats on day 4 after transplantation did not induce transient rejection responses in long-term survival recipient LEW rats, a finding that differed from the results of experiments using normal WF liver homogenates. However, challenge with grafted WF liver homogenates, similar to those of normal LEW liver homogenates, induced rejection responses in long-term survival recipient WF rats with LEW liver allograft. Flow cytometric analysis confirmed that most of nonparenchymal cells in the grafted WF liver were recipient (LEW) genotype. These observations showed that the deletional mechanism of effector T cells also is observed in this setting, and professional donor antigen-presenting cells are replaced by those of recipient genotype within the graft during the early phase of transplantation.

Mitochondrial respiratory function and antioxidant capacity in normal and cirrhotic livers following partial hepatectomy.

For many liver malignancies, major hepatectomy is the usual therapy. Although a normal liver has a tremendous capacity for regeneration, liver hepatectomy in humans is usually carried out on a diseased liver and, in such cases, liver regeneration takes place in a cirrhotic remnant. Mitochondrial function in cirrhotic livers shows a variety of changes compared to control livers. This study investigated how mitochondrial respiratory function and antioxidant capacity change following partial hepatectomy of cirrhotic livers, because liver regeneration requires greater energy demands and control of oxidative stress. Cirrhosis was induced in male Wistar-Furth rats by administration of thioacetamide. NADH-cytochrome c reductase activity, mitochondrial glutathione peroxidase activity and mitochondrial GSH levels were all significantly lowered in cirrhotic livers and in the cirrhotic remnants up to 72 h after 70% hepatectomy when compared to the corresponding controls. Lower respiratory control ratios with succinate as substrate were also observed from 6 to 48 h post-hepatectomy. At 24 h post-hepatectomy, higher levels of lipid peroxidation were observed. We conclude that, compared to the controls, cirrhotic livers have diminished oxidative phosphorylation capabilities due to changes in NADH and FADH(2)-linked respiration as well as impaired antioxidant defenses following partial hepatectomy. Both of these factors, if critical, could then impede liver regeneration.

Deferoxamine Reduces Cold-Ischemic Renal Injury in a Syngeneic Kidney Transplant Model

In cell-culture models, addition of deferoxamine (DFO) to University of Wisconsin Solution (UW solution) reduces cold-storage injury. The efficacy of DFO was therefore tested in a kidney transplantation model employing inbred Wistar Furth rats. Donor left kidneys, cold stored for 18 h in UW solution with or without 0.125 mM or 0.625 mM DFO were transplanted to the recipients' left renal fosse. Deferoxamine dose-dependently and significantly increased glomerular filtration rate (GFR) and renal blood flow (RBF), and suppressed renal F2-isoprostanes (vasoactive lipid peroxidation products) and apoptotic and necrotic injury 3 days post-transplantation. In a second set of similar experiments, the remaining native kidneys of the recipient rats were removed on day 7 of transplantation. Transplanted kidneys' function assessed by serum creatinine was 75% higher in the cold-stored transplanted kidneys treated with DFO compared with untreated kidneys. Moreover, the DFO treatment was attended by a significant reduction in apoptotic and necrotic tubular injury. Thus, our consistent findings from two sets of studies in a transplant model suggest that a simple strategy of including DFO in the cold-storage solution reduces cold ischemia-associated renal transplant damage and improves renal function. Our findings have potentially important ramifications for cold preservation of kidneys, and possibly other organs, in clinical transplantation.

Protection of wistar furth rats from chronic renal disease is associated with maintained renal nitric oxide synthase.

Wistar Furth (WF) rats do not develop renal injury after severe reduction of renal mass. Because clinical and animal studies suggested that nitric oxide (NO) deficiency occurs and may contribute to chronic renal disease (CRD), the status of the NO system in WF versus Sprague Dawley (SD) rats was examined with the 5/6 renal ablation/infarction (A/I) model of CRD. Eleven weeks after A/I, SD rats developed proteinuria, severe kidney damage, decreased renal function, and marked decreases in total and renal NO synthase (NOS), specifically neuronal NOS. In contrast, WF rats exhibited elevated baseline and maintained post-A/I total NO production, with no decrease in renal cortex NOS activity despite a decrease in remnant neuronal NOS abundance. When low-dose chronic Nomega-nitro-L-arginine methyl ester treatment was added for WF A/I-treated rats, rapid progression of CRD was observed. In conclusion, elevated NO production in WF rats was associated with protection from the progression of CRD after renal mass reduction. The protection might be attributable to greater total and renal NO-generating capacity and increased nephron number, compared with SD rats. NOS inhibition rendered WF rats susceptible to progression, suggesting a possible critical threshold for NO production, below which renal injury occurs.

Long-term acceptance of composite tissue allografts through mixed chimerism and CD28 blockade.

Clinical composite-tissue (hand) transplantation between genetically disparate individuals currently requires potent, nonspecific immunosuppressive agents that are neither completely successful in preventing acute episodes of rejection nor free from complications. The reliance on long-term immunosuppression has prompted this study to achieve donor-specific transplantation tolerance in adult recipients using a nontoxic, nonmyeloablative protocol. Fully mismatched, 4- to 6-week-old ACI (RT1Aa) and Wistar Furth (WF) rats were used as donors and recipients, respectively. Recipients were administered CTLA4-Ig at 2 mg/kg per day (alternate days) in combination with tacrolimus at 1 mg/kg per day (daily) from day 0 through day +10, antilymphocyte serum at 10 mg at day +10 (single dose), and total-body irradiation t 300 cGy (day 0) before bone-marrow transplantation (BMT) (day 0) with 100 x 10(6) T-cell-depleted bone marrow cells. Hindlimb transplants were performed 4 weeks postBMT. Multilineage donor hematopoiesis was determined pre- and posttransplant using flow cytometry. In vitro T-cell responses were evaluated by mixed lymphocyte reactivity assays. CD28 blockade in a transplant model of mixed chimerism effectively aborts T-cell clonal expansion in vitro and in vivo, inhibits the development of acute and chronic rejection of vascularized hindlimb allografts in rats (ACI limbs to ACI-->WF chimeras, n=5; WF limbs to ACI-->WF chimeras, n=4), and subsequently leads to long-term survival of allogeneic skin grafts (n=9). Third-party (F344, n=4) transplants were uniformly rejected within 14 days posttransplant. Multilineage donor hematopoiesis was demonstrated pre- and posttransplant. Donor chimerism, present postBMT, increased throughout the study (pretransplant range 2-28%, mean 17%; posttransplant range 5-49%, mean 34%). Transplant recipients maintained full reactivity to respond to third-party antigens without harmful manifestations of graft-versus-host disease. Although efforts have been made to induce tolerance to composite tissue allografts in adult recipients, thus far, none have succeeded without toxic, myeloablative host preconditioning. Our demonstration that tolerance can be achieved with minimal preconditioning provides a rationale for application to large animals and humans and suggests that although composite tissue allografts may have a significant skin component (and are therefore felt to be highly antigenic), protocols used to induce tolerance to organ transplants may be equally applicable to composite-tissue allotransplantation.

Cyclin D1 is necessary but not sufficient for anchorage-independent growth of rat mammary tumor cells and is associated with resistance of the Copenhagen rat to mammary carcinogenesis.

To identify genes associated with the resistance of Copenhagen (Cop) rats to mammary carcinogenesis, we infused a retrovirus harboring v-Ha-ras directly into the main mammary ducts of resistant F1 rats from a cross between Cop and susceptible Wistar Furth (WF) rats. Adenocarcinomas formed in approximately 50% of infused glands. Cell lines derived from these tumors were clonal, but did not share a common viral integration site, suggesting that a high level of v-Ha-ras expression was able to overcome resistance in the F1 rats. Some of the cell lines were able to grow in soft agar, but a significant number did not display anchorage-independent growth. These growth characteristics were independent of v-Ha-ras expression levels. The ability to grow in soft agar was associated with the size of tumors induced by injecting the cells into nude mice, and showed a striking positive association with the expression of cyclin D1. Furthermore, while resistance to anchorage-independent growth was fully overcome by transfection of cyclin D1 in some clones, in the others the effect was partial. A similar pattern of cyclin D1 upregulation and growth in soft agar was also observed when the cells were transfected with an active form of beta-catenin. Hybrid cells from the somatic fusion of an anchorage-dependent to an anchorage-independent clone did not grow in soft agar. These results suggest that while a high expression level of cyclin D1 is necessary for anchorage-independent growth in all clones, it is not sufficient for full growth capacity in soft agar, raising the possibility that the loss of a tumor suppressor gene in the cell lines is required to fully confer anchorage-independent growth. Our anchorage-dependent and -independent rat mammary tumor-derived cell lines may recapitulate the resistance and susceptibility of Cop and WF rats, respectively, to mammary carcinogenesis that could facilitate the identification of breast cancer susceptibility genes.

A rat model of syngeneic bone marrow transplantation during breast cancer therapy.

The purpose of this study was to develop a breast cancer model in rats, in which myeloablative chemotherapy and syngeneic bone marrow transplantation (SBMT) could be evaluated systematically for therapeutic effect. The Wistar-Furth (WF) DMBA-4 breast cancer cell line transplanted into naive WF rats produced rapidly growing tumors that were lethal within 2 months. SBMT was performed following preparation with a regimen (Bu-Cy), consisting of busulfan 16 mg/kg by gastric gavage on days -3 and -2 followed by 250 mg/kg of cyclophosphamide i.p. on day -1. Marrow was prepared from the femurs of donors and infused i.v. into the recipient on day 0. In all, 15 rats treated with Bu-Cy without marrow died, while 22 of 25 transplanted rats survived. In total, 16 rats with measurable tumors showed tumor responses following transplantation, but tumors recurred and survival was minimally prolonged. Of nine rats transplanted before clinical tumors were detected, five became long-term survivors that resisted further tumor challenge. It was concluded that the DMBA-4 breast cancer in WF rats could serve to evaluate SBMT following myeloablative doses of chemotherapy at various tumor loads. At large tumor loads therapy was not curative, but at low tumor burdens cures were possible and resistance to subsequent tumor challenge was demonstrated. The model may be useful for further studies of stem cell infusion in rodent tumor systems.

Altered response in renal blood flow and oxygen tension to contrast media in diabetic rats.

To investigate the effect of the contrast medium (CM) iopromide on renal microcirculation and oxygen tension in non-diabetic control and streptozotocin-diabetic Wistar Furth rats. Oxygen tension was measured with Clark-type microelectrodes and blood flow with laser-Doppler flow probes. In order to differentiate between an acutely increased blood glucose concentration and a long-term diabetic state, some of the non-diabetic control rats were intravenously infused with glucose. CM decreased the medullary oxygen tension in control (non-diabetic normoglycemic) rats ( approximately 35%) but not in diabetic rats. Medullary blood flow in control rats increased after CM administration and remained elevated, while it was unchanged in the diabetic rats. In response to CM, glucose-infused control rats responded similarly to control animals in medullary oxygen tension, but similarly to diabetic rats in medullary blood flow. Contrary to in control rats, medullary oxygen tension was unchanged in diabetic animals after CM administration. Streptozotocin-diabetic rats have an altered response to intravenous injection of the CM iopromide compared to non-diabetic rats. The unaltered medullary oxygen tension, seen in the diabetic group after injection of CM, suggests that non-hemodynamic mechanisms are responsible for the increased frequency of renal failure commonly seen among diabetic patients.

Composite tissue allotransplantation in chimeric hosts: part I. Prevention of graft-versus-host disease.

Mixed allogeneic chimerism (MAC) has been shown to induce tolerance to composite tissue allografts (CTA). However, transplantation of unmanipulated donor-specific limbs results in severe graft-versus-host disease (GVHD). This suggests that nontolerant mature donor-derived cells in the CTA may affect the stability of chimerism, potentially resulting in GVHD. The aim of this study was to develop an approach to study and prevent GVHD in a mixed chimeric-rat hind-limb transplantation model. [ACI-->WF] chimeras received a limb from Wistar Furth (WF) (syngeneic), Fisher (third-party), or ACI (irradiated [1,050 cGy] or nonirradiated) rats. In vitro tolerance was assessed using mixed lymphocyte reactivity (MLR) assays at the time the animals were killed. [ACI-->WF] chimeras with greater than 85% chimerism exhibited rejection-free survival of donor-specific hind limbs. However, 100% of these animals developed lethal GVHD 22.4+/-2.8 days after limb transplantation. [ACI-->WF] chimeras that underwent transplantation with irradiated ACI or syngeneic WF limbs showed no signs of rejection or GVHD at 5 months. Nonchimeric and third-party controls rejected limbs within 10 days. Conditioning of the host WF rats with 950 cGy of irradiation (sublethal, myeloablative) led to high levels of MAC without GVHD. The mature T-cell content of nonirradiated donor (ACI) limbs was sufficient to induce lethal GVHD in 100% of tolerant mixed chimeric [ACI-->WF] hosts. Irradiation of donor limbs before transplantation resulted in long-term donor-specific tolerance and prevented GVHD. These data demonstrate that (1) established chimeras could be susceptible to GVHD caused by immunocompetent donor cells transferred with the hind limb, and (2) inactivating these cells with irradiation prevents GVHD and destabilization of chimerism, and permits rejection-free graft acceptance.

Chronic allograft nephropathy is prevented by inhibition of platelet-derived growth factor receptor: tyrosine kinase inhibitors as a potential therapy.

Chronic allograft nephropathy (CAN) is the primary reason for late allograft loss in kidney transplantation, and currently there is no treatment available for it. Platelet-derived growth factor (PDGF) is suggested to be a major mitogen mediating mesenchymal cell proliferation in CAN. It has been shown that PDGF is already induced at acute renal allograft rejection, indicating a link between acute rejection and subsequent development of CAN. However, the definite effect of PDGF on the pathogenesis of CAN is still unknown. We investigated the role of PDGF in CAN by inhibiting PDGF by imatinib (STI571), a selective PDGF receptor tyrosine kinase inhibitor. Kidney transplantations were performed from Dark Agouti (DA) to Wistar-Furth rats, and syngenic control transplantations were performed from DA to DA rats. All allograft recipients were immunosuppressed with cyclosporine A (1.5 mg/kg/day subcutaneously). One group of the animals was also treated with imatinib (10 mg/kg/day orally). Serum creatinine levels and cyclosporine A concentrations were measured once per week until the animals were killed. Grafts were harvested 5 and 90 days after transplantation for histology and immunohistochemistry. Only very few histologic chronic changes, similar to syngenic grafts, were seen in imatinib-treated allografts compared with control allografts. Creatinine values of imatinib-treated allograft recipients and infiltration of inflammatory cells, PDGF ligand, and receptor induction were also at the same level as in syngenic grafts. Our results demonstrate that imatinib prevents CAN almost completely, indicating that PDGF plays an important role in its pathogenesis. On the basis of our findings, imatinib could be a potential intervention in preventing CAN in clinical kidney transplantation.

Angiopoietin-1 protects against the development of cardiac allograft arteriosclerosis.

Angiopoietin (Ang)-1 is an angiogenic growth factor that counteracts the permeability and proinflammatory effects of vascular endothelial growth factor and other proinflammatory cytokines. Recently, we demonstrated that vascular endothelial growth factor enhances cardiac allograft arteriosclerosis. Here, we studied the roles of Ang1, its natural antagonist Ang2, and their receptor Tie2 in rat cardiac allograft arteriosclerosis. Heterotopic cardiac allografts and syngrafts were transplanted from Dark Agouti (DA) to Wistar-Furth rats and from DA to DA rats, respectively. Immunohistochemistry disclosed that only a few mesenchymal cells expressed Ang1 in normal hearts and syngrafts, whereas no immunoreactivity was found in cardiac allografts undergoing chronic rejection. Ang2 and Tie2 immunoreactivity was induced mainly in capillaries and postcapillary venules in chronic allografts when compared with syngeneic controls, but no immunoreactivity was found in arterial endothelium. Intracoronary perfusion of cardiac allografts with a clinical-grade adenoviral vector encoding human Ang1 (Ad.Ang1) protected against the development of allograft arteriosclerosis. Ad.Ang1 perfusion reduced Ang2 expression in microcirculation, the numbers of graft-infiltrating leukocytes, and the level of immunoactivation and interstitial fibrosis, as well as both the incidence and intensity of intimal lesions. Ad.Ang1 perfusion also increased CD34+ stem cell counts in peripheral blood. Our findings suggest that the antiinflammatory properties of Ang1 may offer an entirely new therapeutic approach to prevent cardiac allograft arteriosclerosis.

Myocyte production of nitric oxide in response to AChR-reactive antibodies in two inbred rat strains may influence disease outcome in experimental myasthenia gravis

In an attempt to identify mechanisms that explain the difference in susceptibility of two rat strains to the induction of experimental autoimmune myasthenia gravis (EAMG), acetylcholine receptor (AChR)-reactive antibodies were tested for their ability to up-regulate levels of inducible nitric oxide synthase (iNOS) in skeletal muscles of disease-sensitive Lewis rats and disease-resistant Wistar Furth (WF) rats. Initially, the WF muscle cell line, WE1, appeared to be more sensitive to antibody-stimulated iNOS induction and NO production than did the Lewis muscle cell line, LE1. Next, AChR-reactive antibody induced widespread iNOS production in skeletal muscles of WF rats, while iNOS production in muscles of Lewis rats was much less pronounced. Finally, inhibition of iNOS activity by administration of a specific iNOS inhibitor resulted in increased susceptibility to the induction of impaired muscle function in EAMG-resistant WF rats. It is speculated that nitric oxide production plays a protective immunomodulating role in WF rats.

Potential importance of glomerular citrate synthase activity in remnant nephropathy

Aldosterone fosters progressive renal injury, but the mechanism is unknown. Both Wistar-Furth rats, which are resistant to aldosterone actions, and adrenalectomized Sprague-Dawley rats, which lack aldosterone, are characterized by resistance to remnant nephropathy and by reduced whole kidney citrate synthase activity. Increase in citrate synthase activity is a well-characterized, specific renal response to aldosterone. Therefore, we performed experiments to test the hypothesis that enhanced citrate synthase activity contributes to remnant nephropathy. Rat models included Wistar (control for Wistar-Furth), Wistar-Furth (resistant to aldosterone), Sprague-Dawley (normal), adrenalectomy (lacking aldosterone), and 5/6 nephrectomy (renal injury). Glomeruli were obtained by differential sieving. Citrate synthase activity was determined spectrophotometrically. Binding characteristics of cytosolic mineralocorticoid receptors were determined by equilibrium competition binding between tritiated and unlabeled aldosterone. Gene sequencing was performed with reverse transcription-polymerase chain reaction (RT-PCR) and fluorescent dye terminators. In glomeruli isolated from adrenalectomized Wistar rats with intact renal mass, aldosterone stimulated a threefold increase in citrate synthase activity; this stimulation was not observed in glomeruli from Wistar-Furth rats. Similarly, citrate synthase activity in glomeruli isolated from adrenally intact Sprague-Dawley rats was 65% greater than that from adrenalectomized Sprague-Dawley rats. Compared to sham surgery, subtotal nephrectomy resulted in 100% greater glomerular citrate synthase activity in Sprague-Dawley rats. In Wistar-Furth rats, mineralocorticoid receptor binding was not reduced, and mutations in the mineralocorticoid receptor DNA binding segment were not found. Citrate synthase activity is elevated in remnant glomeruli, and experimental models characterized by reduced glomerular citrate synthase activity (Wistar-Furth rats, adrenalectomized Sprague-Dawley rats) are protected from remnant nephropathy.

Intrathymic inoculation of donor bone marrow induces long-term acceptance of lung allografts

BackgroundWe investigated whether intrathymic inoculation of donor bone marrow at the time of transplantation induced long-term acceptance of lung allografts. MethodsFour- to-six-week-old August Copenhagen Irish (ACI) and Wistar Furth (WF) rats were used as donors and recipients, respectively. After being inoculated intrathymically with either donor-specific (ACI) or third-party (F344) bone marrow (2.0 × 107 cells/lobe), the recipient (WF) animal received a left lung transplant from an ACI donor. A short course of tacrolimus (1 mg/kg per day for 5 days) was administered. Animals were sacrificed at timed intervals after transplantation, and rejection was graded on a scale of 0 (none) to 4 (severe). ResultsAt 28 days, animals receiving donor-specific bone marrow have lower (p < 0.01) median rejection grade (MRG = 0.25; n = 6) than those receiving third-party bone marrow (MRG = 3; n = 6) and controls (no bone marrow; MRG = 2.5; n = 6). Animals receiving intrathymic donor bone marrow accepted lung allografts up to 380 days with minimal rejection (MRG = 2; n = 6). Long-term lung recipients also accepted a challenging donor-specific heart graft (n = 4) for more than 150 days. In mixed lymphocyte reaction assays, T lymphocytes of WF recipients that had received intrathymic bone marrow (from ACI donor) exhibited low response (similar to self antigens) to donor (ACI) cells, but reacted strongly (five times higher) to third-party (F344) cells. ConclusionsIntrathymic inoculation of donor bone marrow at the time of transplantation along with a short course of tacrolimus induces long-term acceptance of lung allografts in rats. This simple approach of tolerance induction may have clinical application.

Effects of the contrast medium iopromide on renal hemodynamics and oxygen tension in the diabetic rat kidney.

We investigated the effects of the contrast medium (CM) iopromide on regional renal blood flow and oxygen tension (pO2) in the streptozotocin (STZ)-induced diabetic Wistar Furth rats. In normoglycemic rats, CM injection induced a transient decrease followed by an increase in renal cortical blood flow (CBF), whereas CBF increased directly in the diabetic animals. Renal outer medullary blood flow (OMBF) increased in controls, while it decreased in the diabetic animals following CM injection. In control rats a marked initial decrease in OM pO2 following injection of CM was observed. In animals diabetic for 4 weeks only a slight decrease was seen, whereas in 9-week diabetic animals a persistent increase was recorded. An altered oxygen tension and hemodynamic response to CM was found in diabetic rats. If these disturbances may contribute to the development of renal dysfunction by CM in the diabetic rat kidney remains to be elucidated.

Reversal of Diabetes in the Rat by Injection of Hematopoietic Stem Cells Infected with Recombinant Adeno−Associated Virus Containing the Preproinsulin II Gene (Rita Shah et al., Indianapolis, Ind.)

Aim: To study the effect of injecting hematopoietic stem cells containing the preproinsulin gene II (rI₂) via recombinant adeno-associated virus (rAAV) into normal and streptozotocin-diabetic rats. Methods:rI₂was transfected into rat hematopoietic stem cells using rAAV vector. Stem cells were injected by intravenous route into normal and STZ-induced diabetic rats to study blood sugar and expression of rI₂in various tissues. The pLP-1 recombinant plasmid containing rI₂(vLP-1) was engineered as previously described. Bone marrow from female Wistar-Furth rats was enriched for stem cells by using plastic adherence and monoclonal antirat CD3 and CD45 RA to deplete T and B cells. The remaining cells were exposed to vLP-1 (multiplicity of infection MOI = 50:1 or 100:1) for 2 h. Approximately ten million exposed stem cells were injected by intravenous route into each animal; there were four groups: normal animals at MOI 50:1 (group 1) or MOI 100:1 (group 2); group 3 animals (n = 9) were streptozotocin-induced diabetic animals at MOI 100:1. Animals that showed reversal of diabetes from group 3 were sacrificed for study of gene expression at weeks 1, 2, and 6, respectively. Control diabetic animals did not receive stem cells or virus constituted group 4. Expression of rI₂was analyzed by RT-PCR and Southern analyses. Results: Despite introduction of insulin gene, groups 1 and 2 had blood sugar concentrations that remained within normal levels, while 3 of 9 animals in group 3 showed reversal of diabetes; using RT-PCR, group 1 expressed rI₂in liver, spleen, thymus, brain, and heart at week 1 only. In group 2, rI₂ was seen in the thymus up to 6 weeks; in diabetic animals (group 3) rI₂ was seen in liver, bone marrow, spleen, thymus, and peripheral blood lymphocytes at week 2 and in thymus and lymphocytes at week 6. Conclusions: We have shown that (1) rAAV is a useful vector for transferring rI₂into rat hematopoietic stem cells; (2) normal animals remained euglycemic after injection of stem cells containing rI₂despite identification in various tissues suggesting autoregulation, and (3) short-term reversal of diabetes was achieved in some animals by injection of stem cells containing rI₂.

Low-dose immunosuppression in a rat hind-limb transplantation model.

Composite tissue allografts (CTAs) offer an alternative to conventional reconstructive methods. However, the toxicity of the drugs that are required to prevent rejection has prevented its widespread clinical application. The purpose of this study was to determine whether a low-dose, corticosteroid-free combination regimen of tacrolimus and mycophenolate mofetil (MMF) would prevent rejection in a rat hind-limb model, with minimal toxic side effects. Three groups were used in this study. In group I, Wistar Furth (WF) rats received a syngeneic WF hind-limb. In groups II and III, WF rats received an ACI hind-limb. The latter were treated with tacrolimus-MMF. Assessment for rejection, flow cytometry, and mixed lymphocyte reactions was performed. Biopsies were taken regularly and at the time of killing. Combination therapy with low-dose tacrolimus-MMF effectively prolonged CTA survival indefinitely, with minimal side effects. Toxicity associated with immunosuppressive drugs can be avoided in a low-dose combination corticosteroid-free regimen.

Successful Single Donor Islet Allotransplantation in the Streptozotocin Diabetes Rat Model

The objective of this study was to define pretransplant islet culture conditions for optimum tissue engraftment in the rat islet allotransplantation model. Lewis rat islets were cultured in TCM 199/5% fetal calf serum for I day at 37 degrees C, followed by 1 day of culture at 22 degrees C. When islets from single donors were allotransplanted intraportally into single streptozotocin-diabetic Wistar-Furth rats, complete normoglycemia was restored within 1 day after transplantation in seven out of seven rats, and persisted up to immunological rejection about 1 week later. In five out of six rats receiving a posttransplant injection of antilymphocyte serum, plasma glucose was normalized for >100 days. These data demonstrate, for the first time, successful single-donor-to-single-recipient transplantation of allogeneic rat pancreatic islets. Because islet engraftment may still be regarded as a main problem for clinical islet transplantation, the pretransplant islet culture regimen outlined in this article may lead to a more efficient use of donor pancreatic islet tissue in the clinical setting, as well.

The immunomodulatory effect of cryopreservation in rat tracheal allotransplantation

Background Cryopreservation is one solution to the problem of donor organ deficit. To investigate the effect of cryopreservation on tracheal allografts, we performed 2 experiments in rats. Methods In Experiment 1, we assessed second-set graft rejection. Two weeks after primary heterotopic transplantation (Group 1, fresh isografts; Group 2, fresh allografts from Lewis rats; and Group 3, cryopreserved allografts from Lewis rats; n = 5, respectively), each animal underwent secondary heterotopic grafting with isografts and allografts from Lewis and Wistar Furth rats ( n = 5, respectively). Four weeks after the secondary transplantation, all grafts were retrieved for histologic analysis. In Experiment 2, we assessed the long-term results of allograft cryopreservation, without immunosuppression therapy. Six months after transplantation of fresh (Group 4) and cryopreserved (Group 5) allografts, the tracheal segments (each group, n = 5) were histologically evaluated. Results In Experiment 1, only the secondary allografts from Lewis rats in Group 2 did not maintain lumen structure and often showed dislocated or destroyed cartilage. Second-set graft rejection was specifically recognized in Group 2, but not in Group 1 or 3. In Experiment 2, the cryopreserved allografts appeared almost normal and lumen rigidity was preserved 6 months after transplantation. These allografts were superior to the fresh allografts in patency and in cartilage dislocation and mononuclear cell infiltration scores, but not in the viable chondrocyte ratio. Conclusions We conclude that cryopreservation may produce successful long-term results because of its immunomodulatory effect on tracheal allografts.