Onset and evolution of nephropathy in rats with spontaneous diabetes mellitus

The occurrence of renal diabetic complications was studied in diabetic nonobese IIM/FmeSS (eSS) rats. The results were compared with eumetabolic Wistar rats paired by sex and age. Between 6 and 12 months of age, eSS male rats had higher fructosamine values and glucose intolerance as well as increasing proteinuria and uremia. Enhancement in water, calcium and phosphorus fractional excretion with a concomitant lower sodium excretion, was observed from 12 months of age on. 18- and 21-month-old eSS rats exhibited fasting hyperglycaemia and rising values of fructosamine, glucose intolerance and glycosuria. Simultaneously, a notorious worsening of proteinuria as well as alterations in glomerular filtration were verified. Optic microscopy of 12-month-old eSS rat kidneys showed areas of tubular dilatation with protein cylinders. In 21-month-old eSS animals, kidneys appeared overtly damaged. Increased capsular, glomerular and Henle's thin loop diameters were verified in 12- and 21-month-old eSS rats. Glomeruli showed diffuse hypertrophy of mesangial tissue and thickening of the basement membrane. Areas of markedly atrophic and dilated tubules containing acidophilic proteinaceous material were observed. At age of 21 months, kidneys of eumetabolic Wistar control rats presented foci of interstitial and pielic inflammatory infiltrates.     

Volume of the conducting airways calculated from morphometric parameters

A method of calculating the volume of a tree distal to a cut at the origin of a branch, using branching, diameter and length ratios, has been developed. The method was applied to bronchial tree casts from human, dog, sheep, hamster, and rat lungs. It was found that the exponenta in the equation weight=k×diameter ^a is approximately equal to 3.0 in sheep lung casts, as found by Hooper (1977), but it is greater than 3.0 in casts from the other four species.     

Size-dependent morphology of the conductive bronchial tree in four species of myomorph rodents

Size-dependent structural patterns in the conductive bronchial tree of four species of myomorph rodents of different body weight were determined by lung casts. The lungs of the harvest mouse, Micromys minutus, body weight 5–7 g, the house mouse, Mus musculus, body weight 35–45 g, the brown rat, Rattus norvegicus, body weight 200–400 g, and the African giant pouched rat, Cricetomys gambianus, body weight 1,200–1,800 g, were inflated to 20 cm H₂O, frozen, freeze-dried, hardened, and filled with silicone rubber. The casts were pruned, and branching pattern, diameter, and volume of the conductive bronchial tree were determined using a binocular magnifier. All four species have four lobes on the right lung and an undivided left lung, and the central bronchial tree on either side shows an identical monopodial branching pattern. Although the ramification of the central conductive bronchi is not size-dependent, the diameter and volume are. The diameter of the left main bronchus equals 1.24% of body length in Micromys and 0.6% in Cricetomys, and the conductive bronchial tree makes up 13% of the total lung volume in Micromys and 6% in Cricetomys. Relatively wider airways and a decline in airway resistance with declining body mass in small mammals compared to large ones result in a high ventilatory dead space, which is compensated for by a higher breathing frequency. © 1996 Wiley-Liss, Inc.     

A simplified method of casting the macroscopic airways of lungs

A technique for preparing casts of the macroscopic airways of mammalian lungs, which is both simplified and inexpensive in comparison with previous techniques, is described. The models are accurate, durable and flexible, and clearly demonstrate the orientation and branching pattern of the bronchial tree. The nature of the procedure also extends the availability of casts to laboratories or individuals with limited instrumentation and/or funding. Preliminary results using this technique to inject the lungs and certain air sacs of birds are also discussed.     

Serum lipids and lipoprotein composition in spontaneously diabetic BB Wistar rats

Serum lipid and lipoprotein composition in spontaneously diabetic BB Wistar rats, nondiabetic littermates, and control Wistar rats was studied to elucidate diabetes-related abnormalities of lipoprotein composition. Serum total triglycerides and pre-beta-lipoprotein concentrations of insulin-treated spontaneously diabetic BB and nondiabetic littermate rats were significantly higher than those of control Wistar rats. Serum cholesterol and HDL cholesterol concentrations of spontaneously diabetic BB and nondiabetic littermate rats did not differ from controls. Concentrations of very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL) of spontaneously diabetic BB and nondiabetic littermate rats were higher than those of normal rats. With sodium dodecylsulfate-polyacrylamide gel electrophoresis it was observed that the spontaneously diabetic BB and nondiabetic littermate rat VLDL contained higher percentages of apoE relative to total apoC when compared with control Wistar rats. With isoelectric focusing, apoC-II relative percentages in VLDL and HDL of both spontaneously diabetic BB and nondiabetic littermate rats were higher than apoC-II proportions in VLDL and HDL of controls. Apolipoprotein A-I of the control rat HDL showed four isoforms that focused at pI 5.8 (17.3%), 5.75 (30.6%), 5.65 (31.8%), and 5.55 (20.5%); however, the spontaneously diabetic BB and nondiabetic littermate rat HDL apoA-I was mainly represented by two isoforms that focused at pI 5.8 and 5.75. VLDL of both diabetic and nondiabetic BB rats contained higher levels of acidic apoE isoforms compared to their counterparts in control Wistar rats. Although HDL cholesterol concentrations of spontaneously diabetic BB rats remained normal, protein concentrations were higher resulting in a low cholesterol/protein ratio in HDL suggesting that the cholesterol-carrying capacity of spontaneously diabetic BB rat HDL could be less than normal and may be due to an abnormal apoA-I composition. Quantitative alterations of lipid and lipoprotein composition appear in the BB Wistar rat when compared to the Wistar rat, but some of the changes are more pronounced in the spontaneously diabetic BB Wistar rat.     

Quantitative morphological changes in endocrine pancreas of rats with spontaneous diabetes mellitus

The present study describes the cytopathology of the pancreatic islets in 18-old male eSS rats with spontaneous diabetes mellitus as compared to aged-matched normal animals. Light-microscopic immunocytochemical and morphometric techniques were used to study islet-cell populations, while quantitative methods were employed specifically for the analysis of B-cell ultrastructure. The diabetic rats showed disruption of the islet structure and fibrosis in the stroma. The volume density (Vvi) of endocrine tissue and the Vvi and percentage of B cells were diminished, whereas the Vvi of exocrine tissue and the Vvi and percentage of D cells were increased. The number of medium and large islets as well as their mean volume (micron3) decreased in these animals. Pancreatic B cells from eSS rats showed an increase in the Vvi of endoplasmic reticulum, immature secretory granules and lysosomes. Conversely, the Vvi of total secretory granules and microtubules appeared diminished. The current observations contribute to our understanding of this useful animal model of diabetes mellitus, in the attempt to clarify the pathogenesis of the disease.     

Antigen-induced leukotriene release from rat lung in vitro

Chopped lung from inbred hyperreactive rats was challenged with antigen following active or passive sensitization and supernatants were assayed for the presence of leukotrienes (LTs) by radioimmunoassay. Dose-related increases in the release of LTC4- and LTB4-immunoreactive material were obtained with significantly more material being released following passive sensitization. Chromatographic analysis indicated the presence of LTB4, LTC4 and LTE4. When LT release in inbred rats was compared to Sprague-Dawley or Fischer rats, the amounts released were as follows: Inbred greater than Sprague-Dawley greater than Fischer. It was concluded that the release of LTs in the three strains correlated with the degree of non-specific bronchial hyperreactivity.     

The effect of X-radiation on the process of lung clearance after inhalation of bacteria labelled with 3H-thymidine

The respiratory tract is cleared very effectively from the inhaled particles by the ciliated bronchial epithelium, secretion of secretory cells, and specialized phagocytic cells. Within 4 hours about 80% of the inhaled microorganisms are destroyed in the respiratory tract. The rate of removal of the micro-organisms from the lungs and their destruction may play a very important role in preventing infection development. The aim of the present experiment was to study the adverse effect of ionizing radiation on the mechanisms involved in cleaning the lungs. Wistar rats were used for this purpose. They received a dose of 500 cGy. The irradiated animals and the control non-irradiated ones inhaled in a PIANO IV apparatus Staphylococcus epidermidis labelled with tritiated thymidine. Determining the clearance of the bacteria from the bronchial tree and pulmonary tissue an impairment of the mechanisms clearing the lungs in the irradiated group was demonstrated. The effect of radiation may be related to a damage to the alveolar macrophages which are principally responsible for destroying bacteria at the level of pulmonary alveoli. The relatively radiation-resistant ciliated epithelial bronchial cells may have their efficiency reduced owing to disturbances in mucus secretion by the secretory cells.     

Lung tissue engineering and preservation of alveolar microstructure using a novel casting method

Abstract

We used a rat model to decellularize and seed alveolar cells on a three-dimensional lung scaffold to preserve alveolar microarchitecture. We verified the preservation of terminal respiratory structure by casting and by scanning electron microscopy (SEM) of the casts after decellularization. Whole lungs were obtained from 12 healthy Sprague-Dawley rats, cannulated through the trachea under sterile conditions, and decellularized using a detergent-based method. Casting of both natural and decellularized lungs was performed to verify preservation of the inner microstructure of scaffolds for further cell seeding. Alveolar cell seeding was performed using green fluorescent protein (GFP) lung cells and non-GFP lung cells, and a peristaltic pump. We assessed cell seeding using histological and immunohistochemical staining, and enzymatic evaluation. All cellular components were removed completely from the scaffolds, and histological staining and SEM of casts were used to verify the preservation of tissue structure. Tensile tests verified conservation of biomechanical properties. The hydroxyproline content of decellularized lungs was similar to native lung. Histological and immunohistochemical evaluations showed effective cell seeding on decellularized matrices. Enzymatic measurement of trypsin and alpha 1 antitrypsin suggested the potential functional properties of the regenerated lungs. Casts produced by our method have satisfactory geometrical properties for further cell seeding of lung scaffolds. Preservation of micro-architecture and terminal alveoli that was confirmed by SEM of lung casts increases the probability of an effective cell seeding process.     

Pulmonary vascular reactivity in Fischer rats

We previously reported that Fischer (F) rat lungs developed more extensive injury when challenged with oxidants than age-matched Sprague-Dawley (SD) rat lungs. We now describe a reduced pulmonary vascular response to alveolar hypoxia and angiotensin II (ANG II) in F compared with SD rats. The comparative studies were performed with isolated lungs perfused with salt solution or blood, catheter-implanted awake rats, and isolated main pulmonary arterial rings. Isolated lungs from F rats perfused with either blood or salt solution had reduced vasoconstriction in comparison with lungs from SD rats when exposed to alveolar hypoxia or challenged with ANG II. Instrumented awake F rats had a smaller mean increase in total pulmonary vascular resistance (PVR) than SD rats (35 vs. 94 mmHg.min.l-1, P less than 0.05) when challenged with 8% oxygen. The contractile response of isolated pulmonary artery but not thoracic aortic rings to KCl and ANG II was reduced in F compared with SD rats. In addition, F rats exposed to 4 wk of hypobaric hypoxia developed less pulmonary hypertension and right ventricular hypertrophy (when corrected for the hematocrit) than SD rats. We conclude that the oxidant stress-sensitive inbred F rat strain is characterized by a lung vascular bed that is relatively unresponsive to vasoconstricting stimuli. The mechanism underlying this genetic difference in lung vascular control remains to be defined.     

Lung mechanics and connective tissue proteins in diabetic Bio-Breeding/Worcester Wistar rats

We studied lungs of spontaneously diabetic Bio-Breeding/Worcester (BB/W) Wistar rats which resemble human insulin-dependent diabetes mellitus. Compared with the age-matched control group, the body weight of the diabetic rats tended to be smaller and lung wet and dry weight were similar, but lung dry weight, relative to body weight and to lung wet weight, was significantly larger. Both air and saline lung volumes were reduced in the diabetic rats, and volume-pressure (V-P) curves expressed as a percent of maximal lung volume were significantly shifted downward and to the right of those in the control group over the midportion. Total DNA and RNA contents were similar in both groups, whereas protein content and concentration and protein/DNA and RNA/DNA ratios were significantly reduced in the diabetic rats. In contrast, content and concentration of 4-hydroxy-L-proline, elastin, and crude connective tissue were significantly higher in the diabetic group. We conclude that the increase in connective tissue proteins in the BB/W rats is most likely responsible for the shift in the V-P curves.     

Lung morphology in rodents (Mammalia, Rodentia) and its implications for systematics

A new nomenclature of the lung lobes and of the bronchial tree is presented, with which the lungs in 40 species of 11 rodent families are described. Whole, fixed lungs and silicone casts of the bronchial tree are tested for 23 characters, based on the distribution of lung lobes, the number and geometry of first order bronchi, the pulmonary blood supply, and lung symmetry. Ten lung morphotypes are recognized, seven of them representing one or more families: Castor type (Castoridae), Cryptomys type (Bathyergidae), Ctenodactylus type (Ctenodactylidae), Eliomys type (Gliridae), Myocastor type (Myocastoridae), Octodon type (Octodontidae and Echimyidae) and Rattus type (Sciuridae, Muridae pt. and Dipodidae). The Hydromys type is found only in Hydromys chrysogaster (Muridae), while Galea type A and B both appear in Galea musteloides (Caviidae). The data are phylogenetically analyzed by the program PAUP 4.0 using as outgroup Lagomorpha or Insectivora. On the species level, there are no well-resolved cladograms. On the family level, the cladograms do not contradict traditional rodent systematics with one exception: the Caviidae do not fall within Caviomorpha or even within the Hystricomorpha, but form a sister group to Dipodidae (Myomorpha). This appears to be a result of convergence. The lungs of Gliridae are more similar to those of Muridae than to those of Sciuridae. Included in the ingroup, Oryctolagus (Lagomorpha) forms a clade with Caviidae + Dipodidae. Thus, the "Glires hypothesis" is neither supported nor refuted.     

Analysis of genetic diversity and relationships among waxy maize inbred lines in Korea using SSR markers

Information regarding the genetic diversity and genetic relationships among elite inbred lines is necessary to improve new cultivars in maize breeding programs. In this study, genetic diversity and genetic relationships were investigated among 84 waxy maize inbred lines using 50 SSR markers. A total of 269 alleles were identified at all the loci with an average of 5.38 and a range between 2 and 13 alleles per locus. The gene diversity values varied from 0.383 to 0.923 with an average of 0.641. The cluster tree generated using the described SSR markers recognized two major groups at 32% genetic similarity. Group I included 33 inbred lines while group II included 51 inbred lines. The clustering patterns of most of the waxy maize inbred lines did not clearly agree with their source, pedigree or geographic location. The average GS among all inbred lines was 35.7 ± 10.8. Analysis of waxy maize inbred lines collected from Korea and China at 50 SSR loci revealed higher values of average number of alleles (4.9) and gene diversity (0.638) in Korean inbred lines as compared to Chinese inbred lines (3.5 and 0.563, respectively). The information obtained from the present studies would be very useful for maize breeding programs in Korea.     

Vascular responses to agonists in rat mesenteric artery from diabetic rats

The effect of diabetes on vascular smooth muscle function was investigated in the muscular arteries from spontaneously and chemically induced diabetic rats. Isolated ring segments of superior mesenteric arteries from BB diabetic and streptozotocin (STZ)-diabetic rats (12 weeks after onset of diabetes) were used for isometric tension studies. Contractile responses to alpha-adrenoceptor agonists (norepinephrine, methoxamine, phenylephrine, B-HT 920, guanabenz, SKF 89748-A), serotonin, and K+ were significantly higher in STZ-diabetic rat arteries as compared with the controls. In spontaneously diabetic rat arteries only the contractile responses to the putatively selective alpha 2-adrenoceptor agonists, K+ and prostaglandin E1, were significantly increased. pD2 values of the agonists in both groups of diabetic arteries were not significantly different from the respective controls. Nifedipine inhibited all contractile responses in a dose-dependent fashion. The responses to K+ and alpha 2-adrenoceptor agonists were attenuated to a greater extent by nifedipine in both groups of diabetic blood vessels. The calcium channel activator, BAY K 8644, produced a twofold increase in force of contraction in streptozotocin-diabetic and spontaneously diabetic rat arteries as compared with the responses in their respective controls. These results suggest caution in extrapolating all the findings from the streptozocin-induced diabetic model to the spontaneously diabetic model. However, increased activity of calcium channels in vascular muscle cells in both groups of diabetics may be responsible, at least in part, for the increased vascular contractility in diabetes mellitus.     

Cardiac dysfunction in isolated perfused hearts from spontaneously diabetic BB rats

The purpose of this investigation was to examine cardiac function and biochemistry in spontaneously diabetic BB rats, a strain in which diabetes occurs spontaneously and closely resembles insulin-dependent diabetes in humans. The study involved two groups: nondiabetic littermates of BB rats and BB diabetic rats treated daily with a very low insulin dose such that the rats were severely hyperglycemic and hyperlipidemic. The hearts from these two groups were isolated and heart function (using isolated perfused working hearts) and biochemistry were examined 6 weeks after the onset of diabetes. BB diabetic rats exhibited a lower calcium-stimulated myosin ATPase activity and depressed left ventricular developed pressure, cardiac contractility, and ventricular relaxation rates compared with BB nondiabetic littermates. These results suggest that the chronically diabetic state in the BB rat produces cardiac changes similar to those demonstrable after chemical diabetes induced by alloxan or STZ, or that seen during human diabetes mellitus.     

Molecular characterization of endophytic strains of Fusarium verticillioides (= Fusarium moniliforme) from maize (Zea mays. L)

The species Fusarium verticillioides (= F. moniliforme) is often found in maize seeds, constituting an important source of inoculum in the field. Fusarium spp., associated with symptomatic and asymptomatic plants, may be a primary causal agent of disease, a secondary invader or an endophyte. In the present work, endophytic fungi were isolated from two populations of Zea mays (BR-105 and BR-106) and their respective inbred lines. Within different inbred lines of maize, Fusarium was found at a frequency of 0 to 100% relative to the number of total isolated fungi. The frequency with which the genus occurred was practically the same in the two field sites (around 60%). Twenty-one F. verticillioides strains were analysed using the random amplified polymorphic DNA (RAPD) technique, employing 10 random primers. Variability analysis of endophytic isolates via RAPD showed genome polymorphism taxa of species around 60%. Endophytic isolates were clustered by their sites of origin. RAPD analysis clustered the endophytic isolates by their maize inbred lines hosts (Mil-01 to Mil-06), whereas at site A they clustered into two major groups related to the maize gene pool (BR-105 or BR-106 population). All strains isolated from seeds collected in Site A, except strains L9 and L10, were sub-grouped according to maize inbred lines. The analysis showed a discrete sub-grouping at site B. Results obtained here could be explained by a co-evolution process involving endophytic isolates of F. verticillioides and maize inbred lines.     

利用SNP标记及配合力划分超甜玉米自交系的杂种优势群

划分超甜玉米自交系的杂种优势群,筛选配合力高的甜玉米自交系,构建新的杂种优势更强的甜玉米群体,为优良甜玉米的选育提供依据.本研究选用23个自育超甜玉米自交系,采用NC Ⅱ设计得到60(3×20)个组合,研究它们的产量配合力效应并结合56K SNP标记将供试材料进行杂种优势群的划分.结果表明23个自交系的平均杂合率为2....     

Surfactant aggregation in rat lungs: influence of temperature and ventilation

We examined the effect of the ventilatory rate and the temperature of excised lungs and of increased body temperature of anesthetized spontaneously breathing rats on the centrifugal sedimentation of disaturated phosphatidylcholine (DSPC) present in lung lavage returns. More DSPC sedimented from lungs ventilated at low than at high rates, and sedimentation of DSPC and lung volume loss were temperature dependent, 41 greater than 37 greater than 4 degrees C. Most of the noncellular sedimented material was tubular and common myelin; these had diminished ability to lower surface tension rapidly compared with less-aggregated surfactant. More aggregated DSPC accumulated and lung volume decreased more in spontaneously breathing rats anesthetized for 30 min than in rats killed immediately after being anesthetized; these changes were greater after 30 min of anesthesia in hyperthermic rats (40.4 +/- 0.3 degrees C) than in normothermic rats (37.4 +/- 0.1 degrees C). These studies have shown a correlation between the increased accumulation of surfactant as large aggregates and the loss of alveolar stability; however, a cause and effect between these events has not yet been shown.     

Departures from the physical optimality in the bronchial tree of rats (Rattus norvegicus)

We studied the departure from the physical optimality of the bronchial tree of rats using both i) the minimum volume and power and ii) the minimum surface and drag criteria, considering the bronchial junction as the unit study based on Zamir's model for vascular trees. Our results show deviations of the junctions of the bronchial tree from the expected optimums in the proximal airway that can be explained by both, the turbulent or transitional flow regime, and the airway's necessity to distribute its terminal branches in the alveolar surface filling the thoracic volume. The departures of the observed values at the optimum for the minimum volume and power were significantly different than the obtained departure values for the minimum surface and drag criteria. The departure from the optimum was directly related to the diameter of the smallest branch. The slopes of the regressions for the two criteria were different. The regression lines intercept at a bronchial diameter d2 = 0.129 mm. This result agreed with the idea that the tube diameter is limited at small values by the increasing flow resistance with decreasing tube diameter while at large values is limited by the increasing tube volume and dead space with increasing tube diameter.     

Effects of chronic streptozotocin-induced diabetes on the cardiac responses to milrinone

We have studied the effects of milrinone on various cardiac preparations obtained from 6-week streptozotocin diabetic rats. The basal rate of spontaneously beating right atrium from diabetics was significantly lower as compared with controls. Milrinone (5 X 10(-5) to 8 X 10(-4) M) produced a dose-dependent positive inotropic and positive chronotropic effect in left atrium and right atrium, respectively. The positive chronotropic response to milrinone was slightly increased in right atria from diabetic animals. In papillary muscle neither the maximum response nor the pD2 value of milrinone was altered significantly in diabetic animals. The pD2 values of milrinone in right atrium and left atrium were found to be significantly higher in diabetic preparations compared with controls. The data indicate that the responses to milrinone are either unchanged or enhanced in hearts from diabetic animals.