The normoglycaemic biobreeding rat: a spontaneous model for impaired gastric accommodation.

OBJECTIVE: Impaired gastric accommodation is reported in patients with functional dyspepsia (FD). Previous findings in postinfectious patients with FD suggest that low-grade inflammation and dysfunction of nitrergic nerves play a role in impaired accommodation. To date, spontaneous animal models to study the relationship between these changes are lacking. We hypothesise that the normoglycaemic BioBreeding diabetes-prone (BB-DP) rat provides an animal model of inflammation-induced impaired gastric motor function. DESIGN: Control diabetes-resistant biobreeding, normoglycaemic and hyperglycaemic BB-DP rats were sacrificed at the age of 30, 70 and 220 days and gastric fundus tissue was harvested to study nitrergic motor control, inflammation and expression of neuronal isoform of nitric oxide synthase (nNOS) and inducible isoform of nitric oxide synthase (iNOS). Nutrient-induced changes in intragastric pressure (IGP) were measured in normoglycaemic BB-DP rats to study accommodation. RESULTS: No differences in nitrergic function and inflammation were observed between BB-DP and control rats at 30 days. The nitrergic component of the fundic muscle relaxation was reduced in BB-DP rats of 70 and 220 days. This was accompanied by a significant loss of nNOS proteins. IGP significantly increased during nutrient infusion in BB-DP rats of 220 days, indicating impaired accommodation. Infiltration of polymorphonuclear cells, increased myeloperoxidase activity and increased expression of iNOS was observed in the fundic mucosa and muscularis propria of 70-day-old and 220-day-old BB-DP rats. CONCLUSIONS: BB-DP rats of 220 days display altered fundic motor control and impaired accommodation, which is least partially explained by loss of nitrergic function. This may be related to inflammatory changes in the neuromuscular layer, suggesting that normoglycaemic BB-DP rats provide a spontaneous model for inflammation-induced impaired gastric accommodation.

Thymectomy should be the first choice in the protection of diabetes-prone BB rats for breeding purposes.

Diabetes-prone (DP)-BB rats spontaneously develop diabetes and are widely used as an animal model for the study of type 1 diabetes. Since DP-BB rats develop diabetes before or at the time of breeding, such rats used for breeding need to be protected against diabetes development by the transfer of regulatory T cells obtained from diabetes-resistant (DR)-BB rats, by insulin treatment or by thymectomy. Thymectomy of juveniles is not commonly used to protect DP-BB rats, and we investigated whether breeding with thymectomized DP-BB rats was a realistic alternative to the two other methods. No differences in pregnancy rates, numbers of pups per litter or growth rates of pups were found. Moreover, no differences were found in diabetes development in the offspring. Protection of juvenile DP-BB rats by thymectomy is comparable to the other established procedures, is simple and safe, and the rats recover well from the procedure. Breeding with thymectomized animals will reduce the number of animals needed, and it improves the well-being of the animals because it reduces the negative side effects associated with the other procedures such as episodes of hypo and hyperglycaemia. Therefore, although thymectomy is an invasive procedure, we would like to recommend weanling thymectomy as the first choice for the protection of DP-BB rats for breeding purposes.

Effects of recurrent hypoglycemia on brainstem function in diabetic BB rats: protective adaptation during acute hypoglycemia.

To determine whether antecedent recurrent hypoglycemia protects the brain from the adverse effects of a standardized hypoglycemic stimulus, we implanted electrodes in the inferior colliculi of diabetic rats to directly record inferior colliculi auditory-evoked potentials (ICEPs). Awake, chronically catheterized BB rats were studied after 2 weeks of insulin therapy designed to produce either chronic hyperglycemia (hyper-DM, glycated hemoglobin 7.6 +/- 0.4%) or recurrent hypoglycemia (hypo-DM, glycated hemoglobin 6.2 +/- 0.7%), and the results were compared with those observed in nondiabetic rats. When plasma glucose was lowered to and clamped at 2.8 mmol/l, the release of catecholamines was suppressed in the hypo-DM rats (epinephrine: 2.5 +/- 0.4 nmol/l) as compared with hyper-DM and the nondiabetic rats (9.3 +/- 2.3 and 32.7 +/- 6.1 nmol/l, respectively). ICEP latency was significantly delayed in hyper-DM and nondiabetic rats (P < 0.001), but it was unchanged in hypo-DM rats. A more pronounced reduction in plasma glucose (2.0 mmol/l), however, provoked a greater adrenergic response than that seen at 2.8 mmol/l and delayed ICEP latency by 23% in a separate group of hypo-DM animals. These data demonstrate that antecedent recurrent hypoglycemia attenuates the brainstem dysfunction associated with mild to moderate, but not severe, hypoglycemia in diabetic rats. This phenomenon may contribute to the alterations in hypoglycemia counterregulation seen in diabetic patients during intensive insulin therapy.

Pluripotent haemopoietic progenitor cells (CFU-Sd8) in peripheral blood of hereditarily anaemic Belgrade (b/b) rats.

The unique anaemic syndrome of the Belgrade laboratory (b/b) rat is due to an intracellular iron deficiency which is induced by a not yet defined mutation, resulting in impairment of haemopoiesis. We investigated the CFU-Sd8 number and concentration in the peripheral blood of b/b rats to study the relationship between medullary and extramedullary haemopoiesis in this anaemic syndrome. The results show normal concentration of CFU-Sd8 in the peripheral blood of b/b rats. This finding was unexpected in the state of severe anaemia and disturbed growth factor production in b/b rats, where the mobilization of CFU-Sd8 from bone marrow to blood is expected. The results suggest that severe anaemia is not regularly accompanied by the mobilization of pluripotent progenitors from bone marrow to the blood.

Human insulin B chain but not A chain decreases the rate of diabetes in BB rats.

The autoimmune response seen in insulin-dependent diabetes mellitus (IDDM) includes a humoral immune response against human insulin. Early insulin treatment has been used to prevent IDDM in the rodent models of IDDM, and a prevention trial is underway in humans. The metabolic effects of insulin may not be involved in this prevention since, in NOD mice, the use of metabolically inert human insulin B chain was effective. We wished to ascertain whether immunization of diabetes-prone BB/W rats with insulin B chain, A chain, or both could alter the incidence of diabetes. Immunizations began by 30 days of age and the rats were followed until 120 days of age. Only immunization with insulin B chain plus adjuvant was effective in reducing the rate of diabetes. All immunization frequencies were effective, but a significantly lower rate of diabetes was achieved with injections every week. All of the doses tested resulted in significantly lower rates of diabetes. These data confirm in the BB rat model that immunization with insulin B chain in the presence of adjuvant can reduce diabetes incidence. The absence of any metabolic effect of B chain and the requirement for adjuvant suggest that this effect is mediated via modulation of the autoimmune response.

Changes in the aortic endothelium and plasma von Willebrand factor levels during the onset and progression of insulin-dependent diabetes in BB rats.

Endothelial injury has been implicated in the enhanced vascular disease associated with diabetes mellitus. In diabetic humans elevated plasma von Willebrand factor (vWF) levels have been interpreted as an indication of endothelial damage. Using the BB rat as a model of inherited insulin dependent-diabetes mellitus, plasma vWF and aortic endothelial ultrastructural alterations were examined during the first 7 months of diabetes. Total plasma vWF levels were determined by ELISA and vWF multimeric composition by electrophoresis. vWF was identified immunohistochemically. Following the onset of hyperglycemia, there were progressive alterations in aortic endothelial morphology, which were consistent with injury, and aortic intimal thickening was significantly greater in rats diabetic for 7 months compared to age-matched controls. Significant increases in the Weibel Palade (WP) body content of the endothelial cells were observed after 1 week and 2 months of diabetes, but not at later times. Endothelial alterations associated with the possible release of vWF appeared to involve fusion of WP bodies with other vacuoles rather than direct fusion with the cell membrane. Plasma vWF levels in diabetic rats were varied, but were not significantly different from those of control animals and did not correlate with either glucose or insulin levels. The multimeric composition of plasma vWF was also similar at all times in both diabetic and non-diabetic animals. From these observations, plasma vWF levels do not provide an indicator of the endothelial perturbations which occurs in diabetic rats.

Low stress response enhances vulnerability of islet cells in diabetes-prone BB rats.

In islet cells isolated from normal outbred Wistar rats, the known high vulnerability of islet cells toward oxygen radicals or nitric oxide can be abolished by inducing a stress response, such as by heat shock. We show here that islet cells from diabetes-prone BB rats are unable to mount such a protective response. Islet cells from diabetes-prone BB rats without recognizable insulitis were heat stressed. Subsequently, cells were exposed to nitric oxide, to oxygen radicals, or to the beta-cell toxin streptozotocin. While prior heat shock substantially increased the survival of toxin-treated Wistar rat islet cells, no protective stress response was noted for islet cells from diabetes-prone BB rats. Islet cells from diabetes-resistant BB rats were protected by heat stress to the same extent as Wistar rats. A survey of four additional major histocompatibility complex (MHC)-disparate rat strains confirmed the existence of a low and high responder type to stress. Parallel analysis of heat shock protein (hsp)70 induction by Western blot showed a low and high hsp70 response phenotype. A high hsp70 response coincided with a protective stress response. The presence (or absence) of a protective stress response correlated with the preservation (or loss) of intracellular NAD+ in toxin-treated islet cells. The lack of a protective stress response in islet cells from diabetes-prone BB rats, but not in diabetes-resistant BB rats, may promote beta-cell lysis and autoantigen release, and hence could be important for initiation or propagation of the disease process.

Resistance to glomerular injury in the diabetic biobreeding rat.

This study was designed to determine whether the diabetic BioBreeding rat develops significant renal injury following long-term moderate to severe hyperglycaemia. Diabetic and control rats were followed from the onset of diabetes (2-4 months) to 18 months of age. Frank proteinuria and/or albuminuria were always absent. Glomerular filtration rate, measured by inulin clearance (ml min-1 (100 g body weight)-1), was significantly higher in diabetic rats than in controls at 10, 12 and 18 months of age. Advanced glycosylation end-product cross-links assessed by percentage solubility of tail tendon collagen were moderately increased in diabetic compared with control animals. Urinary excretion of advanced glycosylation end-products in unfractionated urine and in urine fractionated for low molecular mass peptides (< 10 kDa) was 11-fold greater in the diabetic rats than in the control group. Urinary excretion of nitric oxide metabolites (nmol NO2- and NO3- (24 h)-1) were significantly (P < 0.05) greater in diabetic rats than in controls after 8 months of age. Mild histopathology resembling human diabetic nephropathy, including increased mesangial volume and glomerular basement membrane thickness, was detected at 18 months of age. The findings of hyperfiltration and mild glomerular morphological changes in diabetic BioBreeding rats are similar to the abnormalities seen in stage 2 human diabetic nephropathy. We hypothesize that two factors which may contribute to the resistance or tolerance to renal injury in the BioBreeding diabetic rat are increased nitric oxide production and the decreased accumulation of advanced glycosylation end-products.

Prevention of type I diabetes with lymphotoxin in BB rats.

We previously reported that nonspecific immunomodulations with a streptococcal preparation (OK-432), an inducer of tumor necrosis factor (TNF), or with recombinant TNF prevented development of insulin-dependent diabetes mellitus (IDDM) in animal models (NOD mice and BB rats). Recently we have further reported that lymphotoxin (LT), a cytokine with functional and structural characteristics similar to those of TNF, also protected NOD mice from diabetes. In this study, we have extended our observation on the LT to BB rats. Male and female BB/Wor rats were treated intraperitoneally with recombinant human LT three times a week from 4 to 11 weeks of age. The cumulative incidence of diabetes by 14 weeks of age was 24/30 (80.0%) in nontreated control rats, whereas it was 10/26 (38.5% vs control, P < 0.01) and 4/29 (13.8% vs control, P < 0.0001) in the rats treated with 1 x 10(3) and 1 x 10(4) of LT, respectively. There was no significant difference in nonfasting blood glucose levels and body weights between nontreated control and LT-treated rats, which were nondiabetic. In the LT-treated rats, intensity of insulitis was significantly reduced in comparison with the nontreated rats. Concanavalin A-stimulated TNF/LT productivity of spleen cells was significantly lower in BB/Wor and BB/Sendai rats than in Wistar rats or other normal rat strains. On the other hand, there was no difference between BB/Sendai and Wistar rats in the in vivo TNF/LT productivity induced with LPS or with IFN-gamma plus LPS, and the TNF/LT productivity of these rats was lower on stimulation with LPS alone, but higher with IFN-gamma plus LPS than the other normal rats. These results indicate that treatment with LT, as well as TNF, modulated autoimmunity and prevented development of IDDM in BB/Wor rats which may be low producers of TNF/LT.

Decreased weight gain in BB rats before the clinical onset of insulin-dependent diabetes.

Inbred specific pathogen-free diabetes-prone (DP) and diabetes-resistant (DR) BB rats were crossed to produce F1 and intercrossed to produce F2 rats. Diabetes segregates in these crosses as a recessive trait on rat chromosome 4. The weight gain of genetically diabetes-prone rats born to F1 healthy parents was studied to avoid effects of maternal diabetes. The weight gain of the F2 rats was initially not different from the F1 parents. The F2 rats later developing diabetes grew in parallel with their non-affected siblings up until the last 9 days before onset. During these 9 days they showed a decreased weight gain compared to their healthy litter-mates regardless of age. We conclude that decreased weight gain precedes the abrupt clinical onset of diabetes in BB rats and that it may be due to processes associated with the selective loss of beta cells.

Synergy between tetrandrine and FK506 in prevention of diabetes in BB rats.

Delayed administration of tetrandrine, a novel broad-spectrum anti-inflammatory agent, to BB rats at a dosage schedule of 20 mg kg-1 day-1 from 79 days of age reduced the cumulative incidence of diabetes from 73.1 to 41.7% (p < 0.01). Brief treatment with the potent immunosuppressive agent FK506 at a dosage schedule of 0.5 mg kg-1 day-1 from 79 days of age for 5 days had no significant effect on the cumulative incidence of diabetes (66.7%, p > 0.1). However, the combination of tetrandrine and FK506 in the afore-mentioned dosage schedules reduced the incidence of diabetes to only 3.6% (p < 0.001). These results suggest that the strong synergy between tetrandrine and FK506 may offer a safe and effective therapeutic strategy for the treatment of patients with recent onset or imminent IDDM.

Histological and immunopathological analysis of recovered encapsulated allogeneic islets from transplanted diabetic BB/W rats.

Allogeneic islets encapsulated in an alginate/poly-L-lysine membrane and transplanted into diabetic BB/W rats resulted in graft failure within 2 weeks of transplantation. Graft failure was associated with a dense pericapsular infiltrate (PCI) that resulted in necrosis of the encapsulated islets. The PCI could be inhibited by immunosuppressive agents, including cyclosporine and dexamethasone, and this resulted in a significant increase in graft survival. Immunopathological characterization of the PCI indicated that there was a predominance of macrophages. T helper cells also appeared to be present in this PCI. Empty capsules were also found to induce a similar PCI that was identical in composition to that found around encapsulated islets. Thus alginate/poly-L-lysine capsules do not appear to be biocompatible and may account for the variable results in islet graft survival found with these capsules.

Erectile and copulatory dysfunction in chronically diabetic BB/WOR rats.

The pituitary-testicular axis, penile reflexes, and copulatory behavior were studied in male BB diabetic rats from 10 to 40 wk of diabetes. Serum testosterone was diminished from 18 to 28 wk of diabetes, and the responses to human chorionic gonadotropin stimulation were blunted. Serum luteinizing hormone (LH) in diabetic rats did not differ from that of the control rats before or after LH-releasing hormone stimulation. Serum follicle-stimulating hormone and prolactin levels were also similar to controls. After 26 wk of diabetes, androgen-sensitive reproductive accessory organs were significantly reduced in size. This also was true for the androgen-sensitive bulbocavernosus and ischiocavernosus muscles. Penile reflexes in these animals from 20 to 32 wk of diabetes were consistently reduced in number and demonstrated prolonged latency. Copulatory behavior was evaluated in these animals at 25 and 28 wk of diabetes and revealed a reduced number of BB diabetic rats showing normal behavior at 25 wk of diabetes. At 28 wk of diabetes, mount latency, intromission latency, ejaculatory latency, and the postejaculatory interval were all prolonged compared with controls. In addition, the number of diabetic animals showing normal behavior was reduced compared with controls. These studies demonstrate that chronically BB diabetic rats develop diminished testosterone and erectile dysfunction that precedes ejaculatory dysfunction in a similar fashion as impotence in diabetic men. We suggest that further studies in this animal model may be critical to the better understanding and treatment of impotence in diabetic men.

Poly I:C induces development of diabetes mellitus in BB rat.

Polyinosinic polycytidilic acid (poly I:C), an inducer of alpha-interferon, accelerates the development of diabetes in diabetes-prone (DP) BioBreeding (BB) rats. This study investigates the effect of administering poly I:C to a diabetes-resistant (DR) strain of BB rats. We compared the incidence of diabetes, the degree of insulitis, the number of NK cells, helper-inducer cells, cytotoxic-suppressor cells, Ia+ T cells, RT6.1+ T cells, and NK cell bioactivity in DR rats treated with saline and with a 5 micrograms/g body wt (poly-5) dose and a 10 micrograms/g body wt (poly-10) dose of poly I:C. The incidence of diabetes was also compared with that of DP rats receiving poly-5. We found that both doses of poly I:C significantly induce the development of diabetes in the DR BB rat. However, treatment of DR rats with the higher dose induces a greater rate of development of diabetes and earlier onset of diabetes than the lower poly-5 dose. The rate of diabetes development and the mean age of onset were similar in poly-10-treated DR and poly-5-treated DP rats. A significant degree of insulitis occurred in all the poly I:C-treated DR rats, even those not developing diabetes. Peripheral blood NK cell number was greater in poly I:C than in saline-treated rats, after 2 wk of treatment and when killed. The percentage of OX19+ peripheral blood mononuclear cells expressing RT6.1 allotype or Ia antigen were similar in poly I:C- and saline-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in bladder function in the one year spontaneously diabetic BB rat.

Micturition characteristics and in vitro urinary bladder function were investigated in insulin-treated spontaneously diabetic BB rats and age-matched non-diabetic controls one year after the onset of diabetes. BB rats weighed less than controls and were hyperglycemic. Diabetic rats consumed larger volumes of water and excreted larger volumes of urine than controls. The frequency of micturitions and the mean volumes of urine excreted per micturition were significantly increased in BB rats compared to age-matched controls. Associated with the micturition changes in the BB rats were significant increases in bladder body mass. Contractile responses of strips from bladder bodies and bases were measured in response to nerve stimulation, carbachol, phenylephrine, ATP, and KCl. No significant differences between controls and diabetics were found in the absolute contractile responses of bladder body strips to nerve stimulation, carbachol, ATP, or KCl. However, if the data were transformed to correct for the increases in tissue mass in the diabetics, there were significant decreases in the responses of bladder body strips from BB rats to carbachol, ATP, and KCl, but not to nerve stimulation. Even after transformation, there were no differences in the responses of bladder base strips to carbachol, phenylephrine, or KCl. The data indicate that significant changes in micturition characteristics are evident one year after the onset of diabetes in the spontaneously diabetic BB rat. These changes are slow in development, since they are absent six months after the onset of diabetes. The changes in micturition and bladder strip contractility are qualitatively similar to, but quantitatively modest in comparison with those caused by streptozotocin-induced diabetes mellitus. The quantitative differences are probably attributable to an ameliorative effect of the insulin received by the BB rat.

Influence of environmental viral agents on frequency and tempo of diabetes mellitus in BB/Wor rats.

Elimination of environmental viruses by cesarean derivation of the University of Massachusetts colony of BB/Wor rats increased the frequency and accelerated the tempo of spontaneous diabetes among diabetes-prone (DP) rats. In contrast, the viral-antibody-free (VAF) environment did not alter the resistance of pre-VAF diabetes-resistant (DR) rats to spontaneous and RT6+ T-lymphocyte-depletion-induced diabetes. Pre-VAF and VAF rats have essentially the same lymphocyte subsets, and VAF-DP rats are susceptible to the adoptive transfer of diabetes and to the diabetes-accelerating effects of polyinosinic-polycytidylic acid injections. These results suggest that the presence of environmental viral pathogens may act to inhibit effector cell function in lymphopenic DP rats while enhancing effector cell activity in nonlymphopenic DR rats.

Variable prevalence of lymphocytic thyroiditis among diabetes-prone sublines of BB/W or rats.

The BB/Wor rat develops spontaneous autoimmune insulin-dependent diabetes mellitus (DM) and lymphocytic thyroiditis (LT). Six different inbred sublines of this rat model have been selected for studying the pathogenesis of DM and, thereby, the prevalence of DM has been carefully monitored and found to be relatively constant. In contrast, we have observed a striking difference in the prevalence and intensity of LT in these six sublines, varying from 100% in NB subline rats to 4.9% in BE subline rats at 105-110 days of age. Excess iodide administration frequently increases the prevalence of LT but did not do so in the two sublines (BB and BE) with the lowest frequency of spontaneous LT. In view of this variable prevalence of LT in the different BB/Wor sublines, it is imperative that investigators studying the pathogenesis and modulation of LT in this rat model select those sublines which express a desired frequency of spontaneous LT.

Breeding and management of BB rats by the aid of the computer program BB RADABA.

In the process of breeding laboratory animals for experimental research a host of data will be produced. Our computer program BB RADABA permits the management of breeding and experimental data as well as planning of experiments and evaluation of experimental results. More than one year of practical work with this program has shown that BB RADABA provides a better survey over the data, moreover it is able to supply objective recommendations of suitable breeding pairs and had the advantage of graphic and statistic data presentation.

Inhibition of type 1 diabetes in BB rats with recombinant human tumor necrosis factor-alpha.

We previously reported that streptococcal preparation (OK-432), which is a TNF inducer, inhibits insulitis and development of autoimmune diabetes in nonobese diabetic (NOD) mice and Bio-Breeding (BB) rats, as animal models of insulin-dependent diabetes mellitus. We have recently shown that recombinant human (h)TNF-alpha also suppresses development of diabetes in NOD mice. In this study we have extended our observation on TNF to BB rats in order to see whether TNF generally inhibits autoimmune diabetes. A total of 5 x 10(4) U of rhTNF-alpha was administered i.p., twice a week to male and female BB rats from 4 to 27 wk of age. The cumulative incidence of diabetes by 27 wk of age in nontreated rats was 36.4% (8/22), whereas that in hTNF-alpha-treated rats was 0% (0/21) (p less than 0.001). The hTNF-alpha-treated rats did not lose body weight and maintained normal blood glucose concentrations. Immunologic and histologic examinations were performed at the end of the experiment. Spleen cell cytotoxicities for NK-sensitive YAC-1 and rat insulinoma (RINm5F) cells in hTNF-alpha-treated rats significantly decreased in comparison with nontreated and nondiabetic BB rats. Intensity of insulitis was also inhibited in hTNF-alpha-treated rats. Interestingly, a huge hepatomegaly and splenomegaly was found in two of the 21 hTNF-alpha-treated rats. The latter consisted of W3/13dull+ and W3/25dull+ cells, which did not exhibit cytotoxicity for either YAC-1 or RINm5F cells. These results indicate that the chronic and systemic administration of TNF has a regulatory role in autoimmune diabetes in BB rats as well as in NOD mice, and that these animals may have a defect in TNF-mediated immunoregulation.