Oral treatment with Hev b 13 ameliorates experimental colitis in mice.

Hev b 13 is an allergenic esterase obtained from the rubber tree Hevea brasiliensis, which has been shown recently to induce human mononuclear cells to release interleukin (IL)-10 in vitro. This immunoregulatory cytokine appears to play an important role in preventing inflammation and mucosal damage in animal models of colitis and in Crohn's disease patients. The aim of this study was to evaluate the therapeutic effect of Hev b 13 in mice with 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis. Two hours following colonic instillation of the haptenizing agent, and daily thereafter for 5 days, Hev b 13 was administered by oral gavage. In mice treated with daily doses of either 0·5 mg/kg or 5·0 mg/kg of Hev b 13, the clinical signs of diarrhoea, rectal prolapse and body weight loss and also histological damage of the distal colon, were reduced significantly, in comparison with water-treated diseased mice. These findings suggest a potent anti-inflammatory activity of Hev b 13; this activity is speculated to be related to its interaction with cells from the immune system.

Effectiveness of iron repletion in the diet for the optic nerve development of anaemic rats.

PURPOSE: To compare the process of myelination in the developing optic nerve (ON) of anaemic rats with the subsequent recovery after being fed an iron-recovery diet. METHODS: In this study, the morphometrical parameters in the ON were assessed by electron microscopy in Wistar rats that were on an iron-deficient diet for 32 days or for 21 days followed by 10 days on an iron-recovery diet. Qualitative and quantitative analyses were performed using representative electron ultramicrographs. Data were analysed by one-way analysis of variance (ANOVA). When differences were detected, comparisons were made using Tukey's post hoc test (P<0.05 was considered to be significant). RESULTS: Qualitative analysis of the ONs in anaemic and recovered animals showed a higher rate of deformed axons and increased lamellar separation in the myelin sheath when compared with the respective control group. The ON of the anaemic group showed a reduced mean density of myelinated fibres when compared with the control group. The fibre area ratio, axon area ratio, and myelin area ratio of large axons/small axons in the ONs of the control group showed the highest values for the myelin areas, axon areas, and total fibre areas. The control group showed a significantly higher myelin sheath thickness when compared with the anaemic and recovered groups. CONCLUSIONS: Our data indicate that iron is necessary for maintenance of the ON cell structure, and that morphological damage from iron deficiency is not easily reverted by iron repletion.

[Focal cerebral ischaemia induced by middle cerebral artery occlusion and the neuroprotective effect of ketoprofen in rats]. / Avaliação da isquemia cerebral focal induzida pela oclusão da artéria cerebral média e a ação neuroprotetora do cetoprofeno em ratos.

Cerebral ischaemia is eventualy observed during neurosurgical procedures and in several clinical entities that may cause severe neurological deficits and even death. Because it is a severe and complex problem, several studies have been done aiming to elucidate the mechanisms of the ischemic phenomenon and aiming to abolish or to diminish its effects, using drugs that protect the neurons from ischaemia-induced damage. Several neurotransmitters play a role in cerebral ischaemia with emphasis to glutamate by its high concentration in the central nervous system. The purpose of this study was to evaluate the effect of focal cerebral ischaemia in the rat through the dosage of the glutamate and morphological findings, and to evaluate a possible protective effect of the ketoprofen to ischemic neurons. Thirty-six rats Wistar were divided into four groups. The first was a control group, the second a sham group and the animals of the third and fourth groups were submitted to induced cerebral ischaemia through selective obstruction of the midlle cerebral artery during 15, 30 and 45 minutes. Animals of the fourth group were previously treated with ketoprofen 15 minutes before the ischaemia. The ischaemia was evaluated through the histopathological examination and through dosage of the extracellular glutamate in vitro. The histopathological examination showed that there was no difference between the animals of the control and of the sham groups. In the animals submitted to ischemia histopathological alterations appeared at 30 minutes and become more intense at 45 minutes of ischaemia. The main findings were interstitial edema, chromatinic disorganization, vacuolization and nuclear desintegration. The animals treated with ketoprofen showed similar alterations, but they were less intense. Decrease in the dosage of glutamate in the parietal cortex of the animals submitted to ischaemia started at 30 minutes and became more intense at 45 minutes of ischaemia and was similar for animals previously treated or not with ketoprofen, indicating that this drug seems not to interfere with the metabolism of the glutamate at the synapses. The morphological findings in the parietal cortex of the animals submitted to ischaemia, previously treated or not with ketoprofen, suggest that this drug has a neuroprotective effect.

Qualitative and quantitative morphology of the vagus nerve in experimental Chagas' disease in rats: a light microscopy study.

Male Wistar rats were inoculated intraperitoneally with approximately 2 x 10(6) Trypanosoma cruzi Y strain blood forms. On days 7, 50, and 185 after inoculation, the animals were killed, and the right cervical vagus nerve was dissected, postfixed in 1% osmium tetroxide, and embedded in epoxy resin (Araldite). Semi-thin transverse sections were stained with 1% toluidine blue, examined by light microscopy, and photographed. An image analysis system was used to measure the area and diameter of each nerve and each fiber visible on the photomicrographs. Inoculated animals killed on days 7 and 185 after inoculation did not present morphologic or morphometric alterations of the vagus nerve. Inoculated animals killed on day 50 after inoculation presented several degrees of structural disorders in the myelin sheaths compared with control animals. The morphometric data demonstrated that the diameter of the myelinated fibers was generally increased in inoculated animals killed on day 50 after inoculation. These results suggest that experimental Chagas' disease in rats causes myelin damage and axonal swelling of the myelinated fibers of the vagus nerve, and that this injury to the vagus nerve may be important for a better understanding of the pathogenic mechanisms of the cardiac and digestive alterations caused by T. cruzi.

Transforming growth factor-beta production during the development of renal fibrosis in rats with subtotal renal ablation.

The histologic changes observed in the remnant kidney model include progressive mesangial expansion with collapse of capillary lumina, interstitial fibrosis and mononuclear cellular infiltration. Transforming growth factor-beta (TGF-beta 1) is an important regulator of extracellular matrix formation. The purpose of this study was to investigate the production and distribution of TGF-beta 1 in the kidney during the development of glomerulosclerosis and renal fibrosis in rats with subtotal renal ablation. Eighty-two female Wistar rats weighing 180-220 g were divided into two groups: 49 rats were subjected to 5/6 renal ablation and 33 to sham operation. Urinary albumin excretion, blood pressure and glomerular filtration rate (GFR) were evaluated after the surgical procedure. We also performed histology and immunohistochemistry and determined mRNA for TGF-beta 1 in the kidneys of these rats 8, 15, 30 and 90 days after operation. The results showed progressively higher immunohistochemical TGF-beta 1 staining in rats with subtotal renal ablation. Cortical renal content of TGF-beta 1 mRNA was also higher in these animals and peaked at day 15. The existence of a temporal association between glomerulosclerosis, interstitial fibrosis and intense mononuclear cellular infiltration on the one hand and higher immunohistochemical TGF-beta 1 staining in the renal cortex on the other show that this polypeptide may contribute to the development of renal fibrosis in this model.

Transforming growth factor-beta in the development of rat diabetic nephropathy. A 10-month study with insulin-treated rats.

We investigated the intrarenal distribution of transforming growth factor-beta 1 (TGF-beta 1) protein and the TGF-beta 1 mRNA levels in the glomeruli and renal cortex of Wistar rats with streptozotocin-induced diabetes before and after the onset of diabetic nephropathy. Monthly urinary albumin excretion, glomerular filtration rate, glomerular volume, renal histology and immunohistochemical reaction for type-I collagen were also studied. The results showed progressively higher glomerular immunohistochemical TGF-beta 1 staining in rats with a diabetes duration of 24 and 40 weeks which was correlated with albuminuria (r = 0.905, p < 0.01) and was temporally associated with the appearance of glomerular deposition of total and type-I collagen. The glomerular content of TGF-beta 1 mRNA was higher in rats diabetic for 20 weeks while lower cortical RNA-TGF-beta 1 levels were found in rats with a diabetes duration of 1-40 weeks. These data suggest that this polypeptide may be an important mediator of diabetic glomerulosclerosis.

Fractionation of Bothrops pirajai snake venom: isolation and characterization of piratoxin-I, a new myotoxic protein.

Whole desiccated venom of Bothrops pirajai was fractionated on a gel filtration (Sephadex G-75) column. Phospholipase A2, arginine esterase and clotting activity profiles of the six fractions (SI to SVI) obtained were determined. Fraction SIV from the gel filtration column was subjected to chromatography on SP-Sephadex C-25. It was resolved into five subfractions (SIV-SP1, to SIV-SP5). Fractions SIV-SP1, SIV-SP2 and SIV-SP3 showed phospholipase A2 activity but, among these fractions, only SIV-SP3 was homogeneous. Induction of myonecrosis by SIV-SP3, SIV-SP4 and SIV-SP5 was demonstrated by their ability to release serum creatine kinase, and for SIV-SP5, to induce histological alterations in the injected mouse muscle. Chemical characterization by determination of mol. wts, isoelectric focusing and direct manual sequencing of the N-terminal region was performed for SIV-SP3, SIV-SP4 and SIV-SP5. When compared with bothropstoxin-I, the myotoxin SIV-SP5 showed the same total number of amino acid residues (121) and constant molar ratio for all but three amino acids. We have named this toxin piratoxin-I (PrTX-I).

Comparison of the effects of lab chow and casein diets based on body and brain development of rats.

1. In order to study the effects of protein quantity and quality on development, Wistar rats were submitted to four different diets during lactation and post-lactation periods. Three isocaloric diets were utilized with 6% (M), 16% (W16) and 20% (W20) of protein (casein), and the fourth diet (C) consisted of a commercial lab chow containing 22% protein. 2. During the lactation and post-lactation periods the body weights of dams and pups were recorded weekly. On the 49th day of age (21 days of suckling and 28 days of ad libitum chow), all animals were sacrificed and the brains removed and weighed. 3. Dams from all groups increased food intake during the lactation period, but dams of the M group consumed a lower amount of diet as compared with other groups. Only the body weight of M dams was affected by diet during the lactation period, but the body weight of pups from the M and C groups was lower than in the other two groups. At 49 days of age C and M pups showed a significantly lower brain weight compared with W16 and W20 pups. 4. Thus, a commercial lab chow diet does not promote normal body and brain development as compared with balanced diets containing 16 or 20% protein. These results emphasize the need for further studies in order to evaluate other biological and behavioral parameters that might be altered by a lab chow diet.

Comparison of the effects of lab chow and casein diets based on body and brain development of rats

1. In order to study the effects of protein quantity and quality on development, Wistar rats were submitted to four different diets during lactation and post-lactation periods. Three isocaloric diets were utilized with 6 per cent (M), 16 per cent (W16) and 20 per cent (W20) of protein (casein), and the fourth diet (C) consisted of a commercial lab chow containing 22 per cent protein. 2. During the lactation and post-lactation periods the body weights of dams and pups were recorded weekly. On the 49th day of age (21 days of suckling and 28 days of ad libitum chow), all animals were sacrificed and the brains removed and weighed. 3. Dams from all groups increased food intake during the lactation period, but dams of the M group consumed a lower amount of diet as compared with other groups. Only the body weight of M dams was affected by diet during the lactation period, but the body weight of pups from the M and C groups was lower than in the other two groups. At 49 days of age C and M pups showed a significantly lower brain weight compared with W16 and W20 pups. 4. Thus, a commercial lab chow diet does not promote normal body and brain development as compared with balanced diets containing 16 or 20 per cent protein. These results emphasize the need for further studies in order to evaluate other biological and behavioral parameters that might be altered by a lab chow diet

Glutamate release in experimental ischaemia of the retina: an approach using microdialysis.

A rabbit eye model of neural ischaemia is described that uses an increased pressure in the anterior eye chamber to block the capillary supply to the retina. A microdialysis probe placed very close to the retinal surface was used to monitor release of amino acids during ischaemia. A large (two- to threefold) increase in the release of glutamate and O-phosphoserine (twofold), but not of six other amino acids monitored, occurred during initial ischaemia. During reperfusion after release of intraocular pressure, much larger (five- to 10-fold) increases in the release of these amino acids were observed. Parallel ischaemic retinal tissue damage was observed. This damage was prevented by ketamine applied locally via a superfusion needle, suggesting that glutamate released during ischaemia, and particularly during reperfusion, was responsible for cell death.

Glomerular permeability to macromolecules in gentamicin-treated rats.

1. To determine the effect of gentamicin on the functional properties of the glomerular barrier, 44 Wistar rats received daily doses of 80 mg/kg body weight for 6 days. Glomerular permeability to neutral dextrans and albumin was evaluated by day 6 and albuminuria was determined on the 1st, 3rd and 5th days of treatment. 2. Treatment induced an intense increase in albuminuria from 74 micrograms/24 h to 11.5 mg/24 h on the 5th day of treatment (N = 11). This increase was associated with the presence of large amounts of albumin in elements of the glomerular filter and in the apical region of the proximal tubular cells (N = 4). Fractional clearances of neutral dextrans having molecular radii in the range of 18-41 A were not significantly different in control (N = 5) and gentamicin-treated rats (N = 7). 3. These results show that gentamicin, a polycation at pH 7.4, produces an increase in the glomerular permeability to negatively charged macromolecules in rats, probably due to interaction of the polycation with negative changes in the glomerular filter.

Glomerular permeability to macromolecules in gentamicin-treated rats

To determine the effect of gentamicin on the functional properties of the glomerular barrier, 44 Wistar rats received daily doses of 80 mg/kg body weight for 6 days. Glomerular permeability to neural dextrans and albumin was evaluated by day 6 and albuminuria was determined on the 1st, 3rd and 5th days of treatment. Treatment induced an intense increase in albuminuria from 74 ug/24 h to 11.5 mg/24 h on the 5th day of treatment (N=11). This increase was associated with the presence of large amounts of albumin in elements of the glomerular filter and in the apical region of the proximal tubular cells (N=4). Fractional clearances of neutral dextrans having molecular radii in the range of 18-41 A were not significantly different in control (N=5) and gentamicin-treated rats (N=7). These results show that gentamicin, a polycation at pH 7.4, produces an increase in the glomerular permeability to negatively charged macromolecules in rats, probably due to interaction of the polycation with negative charges in the glomerular filter

Analysis of urinary albumin excretion in gentamicin-treated rats.

Gentamicin doses of 40 mg/kg body weight/day were administered intravenously to 62 Wistar rats. Nineteen animals were also treated orally with an NaHCO3 solution in place of water. The gentamicin-treated animals showed increased albuminuria immediately after the 3rd day of treatment. The fact that this increase was marked and that it also occurred at similar intensity in the animals treated with gentamicin and NaHCO3 whose tubular lesions were less serious suggests that the proteinuria was of glomerular origin. Albumins with different electrophoretic mobilities were also detected in the urine of these animals. Therefore, the change in electrical charge of the albumin may have contributed to albuminuria and to the nephrotoxicity induced by gentamicin.

Topographic distribution of callosal neurons and terminals in the cerebral cortex of the cat.

This investigation had four goals: First, to study the general topography of the corpus callosum (CC) of the cat. Second, to study the columnar organization of CC terminals and map their banding pattern in the cortex. Third, to examine the relation between CC neuron density and the presence of CC terminal columns. Fourth, to determine whether CC and anterior commissure (AC) neuron distributions are intermixed. Eight adult cats were subjected to partial commissurotomies, and then to large injections of horseradish peroxidase to one cerebral hemisphere. Processing with tetramethyl benzidine revealed retrogradely labelled cells and anterogradely labelled terminals in the cortex of the uninjected hemisphere. The distributions of these cells and terminals were examined by light microscopy and analyzed by computer microscopic methods. The genu of the CC interconnects frontal portions of the cortex, the body interconnects mostly dorsal portions of the cortex, while the splenium interconnects the temporal and occipital cortices. Reconstructions of the CC terminal columns reveal intricate banding patterns in several non-primary areas of the cortex. CC cell density is greater within than outside the terminal columns. CC and AC neurons intermix in the infragranular layers of the neocortex.

Influence of antigen charge in the pathogenicity of immune complexes in rats.

Immune complexes (IC) formed in the presence of excess antigen with native human anionic (AA) or cationic (CA) albumin and anti-human albumin rabbit gamma globulin were administered to 51 female Wistar rats. In animals injected with IC formed with CA, IC deposition in the renal glomeruli (glomerular capillary walls and mesangium) occurred as early as 5 min after injection. These animals also showed slight alterations in renal structure and albuminuria, whereas in the animals injected with IC formed with AA there was no IC deposition in the renal glomeruli nor any alteration in renal structure or albuminuria. The serum complement levels of animals injected with IC formed with CA were significantly lower than those observed in animals treated with similar doses of IC formed with AA. In vitro experiments also showed that the IC formed with CA fixed more complement than those formed with AA.

Distribution of the neurons of origin of the great cerebral commissures in the cat.

Large injections of horseradish peroxidase throughout major portions of the right cerebral hemispheres of four cats revealed extensive distributions of the neurons of origin of the corpus callosum, the anterior commissure and the hippocampal commissures in the uninjected left hemispheres. The distributions of labelled neurons were mapped by semiautomatic computer microscope. The radial and tangential neuron distributions presented here are of a higher density and greater extent than those in previously published studies based on injections of transportable label to more circumscribed areas of the cerebral cortex of the cat. Generally, commissural neurons in the cat were distributed in a bilaminar fashion with supragranular cells more numerous than infragranular cells.

Effect of urinary alkalinization on renal changes produced by cationic albumin.

40-mg doses of human cationic (CA) or anionic (AA) albumin were administered intravenously to 48 normal female rats. Sodium bicarbonate (NaHCO3) was administered to 8 of these animals before CA or AA. Urine alkalinization caused increased renal CA excretion in CA-injected animals, which also showed marked reduction of the intensity of the renal changes produced by CA.

Effects of administration of cationic and native homologous albumin on the kidney.

Different doses of anionic and cationic albumin (CA) were administered intravenously to 29 normal unanesthetized female rats. Administration of 20 and 30 mg of CA produced an increase in urinary excretion of endogenous albumin. Urinary CA levels in the urine samples collected during the first 60 min after injection of 30 mg of CA were higher than anionic albumin levels. A marked increase in the number of endocytic vacuoles, large numbers of images suggesting fusion of vacuoles with lysosomes, extrusion of cell elements into the tubular lumen and tubule rupture were observed in the animals injected with CA. These results show that CA may produce changes in proteinuria and in renal structure.

Influence of autonomic denervation upon the kinetics of the ileal epithelium of the rat.

The effect of autonomic denervation upon the kinetics of the ileal epithelium of rats was studied by tracing 3H-thymidine labelled nuclei and counting mitotic figures and goblet cells. Counts of labelled nuclei and goblet cells provided information about cell migration along the intestinal epithelium. The mitotic index and turnover time of this population were calculated from the mitotic figures counts. Comparing denervated animals with sham operated controls, it was possible to conclude that autonomic denervation, either sympathectomy or parasympathectomy results in a decrease in mitotic activity. As a result of lower mitotic activity in the crypts, the turnover time increases and there is a delay in the migration of cells from the crypts towards the villi. Sympathectomy causes an early short-lived effect upon the kinetics of the crypt cell population, since after 27h there is a tendency to normality. Parasympathectomy produces a slower but continuous decline in mitotic activity.