Electrocardiographical findings of WBN/Kob rats

Electrocardiographical (ECG) investigations were carried out on 4 and 12 week old WBN/Kob and Wistar male rats. In comparison with Wistar rats which showed ECG findings typical of those of normal rats, WBN/Kob rats showed significantly larger QRS complex amplitude, smaller T wave amplitude, longer QRS duration, and longer QT interval at 4 weeks of age. This tendency became clearer at 12 weeks of age. There were no changes in serum inorganic ion concentrations or cardiac histopathology suggestive of these ECG changes in WBN/Kob rats. These ECG findings in WBN/Kob rats are considered to be a hereditary characteristic of the strain.     

Insulin-Induced Hypoglycemic Peripheral Motor Neuropathy in Spontaneously Diabetic WBN/Kob Rats

Intensive insulin therapy can lead to hypoglycemia, with patients sometimes developing hypoglycemic neuropathy. Spontaneously diabetic Wistar Bonn Kobori (WBN/Kob) rats develop diabetic peripheral motor neuropathy characterized by segmental demyelination and axonal degeneration. We examined the short-term effects of hypoglycemia on neuropathic changes in these rats. Spontaneous diabetic WBN/Kob rats received insulin implants for 40 d and were divided into 3 groups based on blood glucose levels: group N, normoglycemic to slightly hyperglycemic (150 to 250 mg/dL); group H, hypoglycemic to slightly hyperglycemic (50 to 200 mg/dL); and group D, nontreated spontaneously diabetic (350 to 420 mg/dL). Conduction velocity was measured in sciatic–tibial motor nerves; these nerves also underwent qualitative and quantitative histomorphologic analysis. Conduction velocity was not significantly different in N, D, and H groups. Morphologic analysis of the sciatic nerves of H rats showed severe changes, including axonal degeneration, myelin distention, and endoneurial fibrosis, that tended to occur in large, myelinated fibers. N and D rats showed relatively mild changes. The degree and distribution of degenerated nerve fibers in H rats were significantly higher than in N and D rats. These results suggest that hypoglycemia of less than 50 mg/dL induced severe peripheral neuropathy. Hypoglycemic lesions differed from the hyperglycemic lesions in diabetic WBN/Kob rats. This rat strain is an appropriate model for investigating the hypoglycemic peripheral neuropathy that can be associated with a diabetic condition.Peripheral neuropathy is a leading complication of diabetes mellitus. Although its exact pathogenesis is not fully understood, chronic hyperglycemia and resultant microenvironmental changes in peripheral nerve tissue contribute to the development of neuropathy.⁵ Therefore, intensive insulin therapy is needed to prevent such complications in patients with type 1 diabetes. However, intensive insulin therapy can lead to hypoglycemia, with patients sometimes developing hypoglycemic peripheral neuropathy.⁸Although experimental hypoglycemic peripheral neuropathy has been studied by using animal models of type 1 diabetes, few studies have included morphologic analyses.^4,7,14,15 These studies showed that hypoglycemia causes axonopathy involving both degenerative and regenerative events. However, hyperglycemic peripheral neuropathy characterized by axonal atrophy has also been induced in diabetic animal models, such that the hyperglycemic changes in these models were similar to hypoglycemic changes. Diabetic WBN/Kob rats spontaneously develop diabetic peripheral motor neuropathy characterized by segmental demyelination and secondary axonal degeneration.^12,13,19 Morphologic changes in diabetic peripheral motor neuropathy are characterized by various degenerative and regenerative changes in myelin sheath, demyelination, and a shift toward axons of smaller diameter. Therefore, WBN/Kob rats may be useful for distinguishing hyperglycemic from hypoglycemic changes. In addition, the threshold of hypoglycemia that induces the morphologic and clinical changes characteristic of peripheral neuropathy in diabetic animals remains unclear.¹⁴ In the present study, we investigated the effects of short-term hypoglycemia on peripheral neuropathic changes in diabetic WBN/Kob rats.     

Effect of gonadectomy on the onset of diabetic syndrome in the female WBN/Kob rats

Almost all the male animals of WBN/Kob rat strain show the diabetic syndrome whereas none of the female animals develop such diseased conditions even at elder age. We investigated the effect of sex hormones on the onset of diabetic syndrome of this rat strain by comparing the results of body weight gain and various clinical tests such as glucose tolerance, serum biochemistry and histopathology of spayed female rats with those of non-treated and sham-operated female animals kept until 88-week-old. Non-treated and sham-operated female animals had shown no abnormal result even at 88-week-old. Spayed female animals began to reveal glucosuria associated with polydipsia and polyuria from 72-week-old, and gradually developed emaciation and cataract. Increased body weight gain, impaired glucose tolerance and lasting hyperglycemia were observed prior to the onset of the symptoms. Pancreatic changes consisted of atrophy of acinar tissue and atrophy or disappearance of islet tissue attributable to clinical data also were detected in spayed female animals. These diabetic syndrome and pancreatic change were analogous to those of aged male WBN/Kob rats but the onset of spayed females was delayed and less severe. Present results suggest that female sex hormones are protective from the onset of diabetic syndrome of WBN/Kob rats.     

Quantitative Trait Locus Analysis for Chronic Pancreatitis and Diabetes Mellitus in the WBN/Kob Rat

A causative gene mutation is still undefined in approximately half of patients with hereditary pancreatitis, and no genetic factor has been identified in most patients with sporadic chronic pancreatitis. To identify a pancreatitis-associated gene, we performed a quantitative trait locus (QTL) analysis for the traits of chronic pancreatitis and diabetes mellitus in WBN/Kob rats. We identified two highly significant QTLs for chronic pancreatitis and/or hyperinsulinemia on chromosomes 7 and X. These QTLs were located on completely different chromosomal regions from those of causative genes that have been reported for human chronic pancreatitis: PRSS1, CFTR, and SPINK1. For these QTLs, prevalences of the WBN/Kob allele significantly increased in the rats with chronic pancreatitis. These findings indicate that chronic pancreatitis in WBN/Kob rats is controlled by multiple genes, and a genetic analysis in WBN/Kob rats might be useful for gene targeting for human chronic pancreatitis.     

Characterization of a novel congenic strain of diabetic fatty (WBN/Kob-Lepr(fa)) rat

The WBN/Kob-Lepr^fa rat is a new congenic strain for the fa allele of the leptin receptor gene (Lepr). Homozygous (fa/fa) WBN/Kob-Lepr^fa rats provide a model of non-insulin-dependent diabetes with obesity. Here, we describe the characteristics of this new animal model in detail. At 7 weeks of age, both male and female obese WBN/Kob rats showed inflammatory cell infiltration of the pancreas that suggested pan-pancreatitis and an abnormal OGTT. At 3 months of age, both male and female obese WBN/Kob rats developed overt diabetes mellitus associated with severe chronic pancreatitis. In contrast, lean female WBN/Kob rats do not develop pancreatitis or diabetes. In WBN/Kob rats, this mutation might promote the onset of severe pancreatitis, leading to the rapid development of diabetes mellitus.     

Effect of sex hormones on the onset of diabetic syndrome in WBN/Kob rats]

The effects of sex hormone on diabetic conditions were investigated in WBN/Kob strain rats, i.e., castrated or spayed, hormone-treated, and non-treated rats. The effects of sex hormone on glycosuria, body-weight change, glucose tolerance and histopathology of the pancreas were compared among these animals. There were no abnormal changes in these parameters in the non-treated females and estrogen-treated males. The glycosuria began to be observed from the age of about 30 weeks in the non-treated group and from the age of 52 weeks in the castrated group. In the female animals, this symptom began to appear from the age of 55 weeks in the testosterone-treated group and from the age of 72 weeks in the spayed group. Before the onset of the diabetic symptoms, glucose tolerance was impaired in these animals. Body weights of the castrated and estrogen-treated males were lower than that of the non-treated males, especially in the estrogen-treated males. Those of the spayed and testosterone-treated females were much heavier than that of the non-treated females. Testosterone treatment accelerated body-weight gain in the spayed female animals. Histopathological examination of the pancreas revealed atrophy of the aciner tissue and atrophy and disappearance of the islet cells similar to those of the non-treated WBN/Kob male rats in the castrated males, spayed females and testosterone-treated females. However, these changes were not observed in the non-treated females or estrogen-treated males. These findings suggest that female hormone suppressed the onset of hyperglycemia along with glycosuria and male hormone accelerates the onset of hyperglycemia in the WBN/Kob rats.     

Secretory apparatus assessed by analysis of pancreatic secretory stress protein expression in a rat model of chronic pancreatitis

Secretory stress proteins (SSP) are a family of proteins including isoforms of pancreatitis-associated protein (PAP) and pancreatic stone protein (PSP/reg). In vitro exposure to trypsin results in the formation of insoluble fibrillar structures. SSP are constitutively secreted into pancreatic juice at low levels. The WBN/Kob rat is a model for chronic pancreatitis, displaying focal inflammation, destruction of the parenchyma and changes in the architecture of the acinar cell; the synthesis and secretion of SSP are also increased. We have investigated the secretory apparatus by SSP immunohistochemistry at the light- and electron-microscopical (EM) levels. Immunocytochemistry of PSP/reg in Wistar control rats reveals low levels, with individual acinar cells exhibiting high immunoreactivity in zymogen granules. PAP is not detectable. In the WBN/Kob rat, PSP/reg and PAP immunoreactivity is markedly increased. Double immunofluorescence for PSP/reg and PAP I or II demonstrates that these proteins colocalize to the same cell. Acinar cells change their secretory architecture by fusion of zymogen granules and elongation of the fused organelles. The immunogold technique has demonstrated an increase of SSP in zymogen granules in WBN/Kob rats. PSP/reg-positive zymogen granules fuse to form elongated structures with fibrillar contents. An extensive PSP/reg-positive fibrillar network is established in the cytosol. Extracellular fibrils have been observed in several ductules. Thus, SSP-derived fibrils form concomitantly with acinar damage in the WBN/Kob rat. Based on the known tryptic cleavage site of SSP, the in vivo generation of fibrils is presumably the result of premature trypsin activation.     

A new diabetic strain of rat (WBN/Kob)

A new, spontaneously occurring diabetic syndrome has been observed in the aged males of an inbred strain of Wistar rats, WBN/Kob. The main clinical sign, glycosuria, was first detected at about 60 weeks of age, and thereafter some animals developed hyperlipidaemia and gradual emaciation. Prior to the onset of glucosuria, male rats showed impaired glucose tolerance after a glucose load at 21 weeks of age. The histopathologic lesions of the pancreas in the diabetic males consisted of multifocal fibrosis, decreased in number and size of islets and atrophy of exocrine tissue. Multifocal inflammatory foci of varying stages were the main pancreatic lesion in prediabetic male rats. This inflammatory change was detected even in 12-week-old rats and tended to occur around the islets. Therefore focal fibrosis and the decrease in the number and size of islets were considered to result from post-inflammatory scarring. The maturity-onset of this syndrome and the impaired glucose tolerance in younger animals suggested that diabetes mellitus of this rat strain is insulin-independent type II. However, the histological lesions of the pancreas were somewhat different from previous reports of both type I and II diabetes mellitus in man and animals.     

The morphological changes of exocrine pancreas in chronic pancreatitis

The following changes were found by either light or electron microscopic observation of the pancreas in spontaneously developed chronic pancreatitis models (WBN/Kob rats, spontaneously hypertensive rats, and rats with common bile-pancreatic duct stones) and in experimental models of chronic pancreatitis (alcoholic pancreatitis, ischemic pancreatitis, and obstructive pancreatitis): 1) the units of lobules, which were constituted by acinar cell deletion, ductular proliferation, and fibrosis; and 2) tortuous or helical ductal channels of pancreatic ducts with periductal fibrosis, which had many crater-like depressions and very long cilia in their inner surface. These are considered to be the results of obstructive pancreatitis, which are caused by the reactions of defensive factors against the increase of pancreatic duct pressure, including the apoptosis of acinar cells, the hyperplasia and hypertrophy of duct cells, a tighter junctional complex of duct cells, and periductal fibrosis.     

Role of fibrosis-related genes and pancreatic duct obstruction in rat pancreatitis models: implications for chronic pancreatitis

Human chronic pancreatitis is characterized by irreversible fibrosis, whereas pancreatic fibrosis in animal models is reversible. In this study, we compare the development of pancreatic fibrosis in the dibutyltin dichloride (DBTC) model, WBN/Kob rats and bile duct-ligated (BDL) rats. DBTC (8 mg/kg) was administered to LEW rats, and the pancreas was histopathologically investigated sequentially. Male and female WBN/Kob rats aged 4, 6 and 8 months were also examined. BDL rats were prepared by ligation of the bile duct at the duodenal portion and sacrificed at 3 or 7 days after ligation. Fibrosis in the DBTC model peaked after 1 week and was limited to the areas around the pancreatic ducts after 2 weeks, and was composed of both type I and type III collagen. In contrast, fibrosis in male WBN/Kob rats peaked at age 4 months, expanded into intralobular area, and was composed of type III collagen. It exhibited almost no type I collagen and a marked tendency to regress. Pancreatic fibrosis in BDL rats was somewhat difficult to induce and required increased stimulation. This suggests that fibrosis in human biliary pancreatitis may gradually form based on weak, continuous stimulation. We conclude that type I collagen may be involved in the progression of irreversible fibrosis. The imbalance between synthesis and degradation of extracellular matrix molecules or degree of stimulation over a certain period may lead to pancreatic fibrosis. Gene expressions of prolyl hydroxylase and tissue inhibitors of matrix metalloproteinase-2 were elevated.     

Effects of Ca2+ withdrawal on diaphragmatic fiber tension generation

The effects of extracellular Ca2+ withdrawal were studied on isolated diaphragmatic muscle fibers and compared with the effects on the papillary, soleus, and extensor digitorum longus (EDL) contractility, using the same in vitro model. Diaphragmatic fibers were obtained from 15 rats, and papillary muscles, soleus, and EDL were obtained from 10 animals. Isometric force generated in response to 1-Hz supramaximal electrical stimulation was measured with a highly sensitive photoelectric transducer. After control measurements, perfusion with a Krebs solution depleted of calcium (0 Ca2+) was started while the fibers were continuously stimulated (4 times/min) and twitches recorded. For the papillary fibers, perfusion with zero Ca2+ was followed by an immediate decrease in twitch tension, complete twitch abolition occurring within 3 +/- 1 min after zero-Ca2+ exposure. Diaphragmatic fibers behaved similarly, although twitch abolition was delayed (10 +/- 3 min after 0-Ca2+ exposure). For the soleus fibers, the twitch amplitude amounted to 38 +/- 10% of control (62% decrease on the average) after 30 min of zero-Ca2+ exposure, no twitch abolition being noted even after 1 h of Ca2+-free exposure. The twitch amplitude of the EDL fibers amounted to 75 +/- 7% of control (25% decrease) after 30 min of zero-Ca2+ exposure. The recovery kinetics for the four fiber types after reexposure to Ca2+-containing solution were also different, with papillary and diaphragmatic fibers recovering completely within 2.5 +/- 0.5 and 4 +/- 0.5 min, respectively. By contrast, neither the soleus nor the EDL showed complete recovery after 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

p38MAPK suppresses chronic pancreatitis by regulating HSP27 and BAD expression

Mitogen-activated protein kinases (MAPKs) are ubiquitous proteins that function in both normal and stress-related pathophysiological states of the cell. This study aimed to analyze the importance of p38MAPK in pancreatic injury using WBN/Kob rats with spontaneous chronic pancreatitis. Male WBN/Kob rats were injected with the p38MAPK inhibitor SB203580, starting at the age of 4 weeks, and sacrificed 6 weeks later. Compared with vehicle-treated rats, p38 inhibitor-treated rats exhibited a significant increase in pancreatic cell death and inflammation as assessed by histologic examination and myeloperoxidase activity, respectively. p38 inhibition decreased the expression of heat shock protein 27 (HSP27), an antioxidant protein, and enhanced accumulation of reactive oxygen species (ROS). In addition, the proapoptotic protein BAD was increased in the pancreas of rats treated with p38 inhibitor. In a pancreatic cell line (PANC-1), HSP27 knockdown augmented reactive oxygen species accumulation and cell death induced by tumor necrosis factor-α plus actinomycin D. In conclusion, p38MAPK suppresses chronic pancreatitis by upregulating HSP27 expression and downregulating BAD expression.     

Morphological changes in the rat exocrine pancreas after pancreatic duct ligation

In the present study, morphological changes of the exocrine pancreas in rats after pancreatic duct ligation were examined with light microscopy (hematoxylin-eosin, TUNEL, and PCNA staining) and scanning electron microscopy in order to elucidate the effects of increased pancreatic duct pressure. On the fifth day after pancreatic duct ligation, ductular proliferation, periductal fibrosis, and disappearance of acini were observed. TUNEL and PCNA staining demonstrated many apoptotic acinar cells and proliferating ductal cells immediately after ligation, which reached a maximal number on the 2nd or 3rd day. Tortuous or helical interlobular pancreatic ducts with inner surfaces containing many crater-like depressions and long cilia were found after ligation. These changes were almost identical to those observed in the pancreatic tissue of model chronic pancreatitis rats, WBN/Kob rats, and stroke-prone spontaneously hypertensive (SHRSP) rats. In summary, the morphological changes observed after pancreatic duct ligation were similar to those of chronic pancreatitis, therefore, the characteristic changes of pancreatic ducts observed in chronic pancreatitis may be caused by increased pancreatic duct pressure.     

The motor units of cat medial gastrocnemius: electrical and mechanical properties as a function of muscle length.

The effects of changing muscle length on the mechanical properties of 89 motor units from adult cat medial gastrocnemius have been studied in eight experiments. Few differences were found between the effects of length on tetanic tension, twitch tension, twitch-tetanus ratio, twitch contraction time, twitch half relaxation time, rate of force development and electrical activity for fast contracting (twitch contraction time less than or equal to 45 msec) and slowly contracting (greater than 45 msec) units. Those differences that did appear did not persist when these two groups were matched by tetanic tension. It is concluded that the biophysical mechanisms responsible for the changes in mechanical and electrical properties with length must be similar for fast and slow twitch units and not related to potential differences in their muscle fiber type. The effects of changing muscle length on the mechanical properties of the eight whole muscles suggest that changes in force output with length are of minor importance during normal movements as the muscle is found to be electrically active over a relatively narrow range of lengths close to the optimum length for tetanus of the whole muscle. The very shortest muscle lengths at which there is only minimal force development are not used in natural movements, while the declining limb of the length tension curve is at muscle lengths beyond the maximum in situ length.     

The Impact of Magnesium on Isometric Twitch Parameters and Resting Membrane Potential of the Skeletal Muscle in Diabetic Rats

To present the relationship between oral magnesium supplementation, blood glucose, and changes in isometric twitch parameters, resting membrane potential (RMP), in the gastrocnemius muscle in diabetic rats. Sixty rats were used in this study. The rats were divided into four groups: control (drinking tap water, Group I, n = 15), control with treated with magnesium sulfate (10 g/L) (Group II, n = 15), diabetic (Group III, n = 15), and diabetic with treated with magnesium sulfate (10 g/L) (Group IV, n = 15). In Group II and IV, the level of plasma magnesium was increased comparing to those of the control group (p < 0.05). Isometric twitch tensions were decreased significantly in the Group III, but Group IV isometric twitch tensions were increased significantly. Group IV RMP values were close to the Group I. Hyperglycemia decreases gastrocnemius muscle isometric twitch tension and increases RMP in diabetic rats. Magnesium treatment can prevent these diabetic complications.     

Endothermic force generation in fast and slow mammalian (rabbit) muscle fibers.

Isometric tension responses to rapid temperature jumps (T-jumps) of 3-7 degrees C were examined in single skinned fibers isolated from rabbit psoas (fast) and soleus (slow) muscles. T-jumps were induced by an infrared laser pulse (wavelength 1.32 microns, pulse duration 0.2 ms) obtained from a Nd-YAG laser, which heated the fiber and bathing buffer solution in a 50-microliter trough. After a T-jump, the temperature near the fiber remained constant for approximately 0.5 s, and the temperature could be clamped for longer periods by means of Peltier units assembled on the back trough wall. A T-jump produced a step decrease in tension in both fast and slow muscle fibers in rigor, indicating thermal expansion. In maximally Ca-activated (pCa approximately 4) fibers, the increase of steady tension with heating (3-35 degrees C) was approximately sigmoidal, and a T-jump at any temperature induced a more complex tension transient than in rigor fibers. An initial (small amplitude) step decrease in tension followed by a rapid recovery (tau(1); see Davis and Harrington, 1993) was seen in some records from both fiber types, which presumably was an indirect consequence of thermal expansion. The net rise in tension after a T-jump was biexponential, and its time course was characteristically different in the two fibers. At approximately 12 degrees C the reciprocal time constants for the two exponential components (tau(2) and tau(3), respectively, were approximately 70.s(-1) and approximately 15.s(-1) in fast fibers and approximately 20.s(-1) and approximately 3.s(-1) in slow fibers. In both fibers, tau(2) ("endothermic force regeneration") became faster with an increase in temperature. Furthermore, tau(3) was temperature sensitive in slow fibers but not in fast fibers. The results are compared and contrasted with previous findings from T-jump experiments on fast fibers. It is observed that the fast/slow fiber difference in the rate of endothermic force generation (three- to fourfold) is considerably smaller than the reported differences in the "phosphate release steps" (> 30-fold).     

Effect of ionophore A23187 on the strength of contraction and transmembrane action potential of guinea pig papillary muscle]

Calcium ionophore A23187 being added at 2.5 mM concentration induced 2--3-fold increase of peak contractile force of isolated guinea pig papillary muscle stimulated with the frequency of 0.2 Hz. The increase of force of contraction is not followed by any change in amplitude and duration of membrane action potential. A23187 decreases testing tension as well as duration of the twitch and increases relaxation rate of the muscle. This phenomenon probably reflects increased capability of sarcoplasmic reticulum in the process of calcium ion accumulation. In the presence of ionophore appearance of shoulder on the rising part of the twitch has been registered. In this case the single twitch being differentiated reveals two positive maxima. Addition of D-600 leads to decrease of action potential duration and eliminates the second part of the twitch, the amplitude and the velocity of the first phase of contraction being without change. It was proposed that the first component of the twitch appearing in the presence of A23187 and insensitive to D-600 corresponds to the release of calcium from some intracellular stores.     

Tension in frog single muscle fibers while shortening actively and passively at velocities near Vu.

Experiments were undertaken to determine the contribution of passive tension to total tension during rapid shortening in a stimulated muscle fiber. Results were obtained by applying shortening movements at constant velocities slightly less than Vu (the velocity of unloaded shortening) to intact twitch fibers isolated from the frog (Rana temporaria). The tension maintained by unstimulated fibers during such shortening movements ("dynamic passive tension") from moderately long lengths was greater than zero but much less than the passive tension measured under static conditions ("static passive tension") at the same lengths. Fibers maximally activated by electrical stimulation and then shortened at the same velocity over the same range of average sarcomere lengths maintained tension that was greater than zero but less than the dynamic passive tension. For average sarcomere lengths up to approximately 3.1 microns, the dynamic passive tension appeared to be substantially abolished by activation. The onset of the apparent disappearance of dynamic passive tension was studied by initiating the stimulation and the shortening movement simultaneously. The resulting tension response exhibited a latency relaxation that was increased in amplitude compared with the isometric case, followed by a brief tension rise, giving way to a steady tension level equal to that expected if stimulation had been initiated well before the release. These changes are qualitatively explained in terms of the establishment of a steady state distribution of deformations of attached cross-bridges.