Cold-acclimation improves cold-tolerance of diabetic rats

1. 1. The aim of these experiments was to study the extent to which previous cold-acclimation improves the cold-tolerance of diabetic rats. 2. 2. Alloxan diabetic rats (fasting blood glucose higher than 200mg/dl) were used in the experiments. 3. 3. In Expt. 1, non-cold-acclimated control and diabetic rats were exposed to cold environment (7-9°C), and the percentage of survival calculated during a 12-day experimental period. In Expt. 2, the rats were previously cold-acelimated before alloxan or saline injection (diabetic and control cold-acclimated rats) and the survival rate was also assessed during a 12-day period in the cold. 4. 4. The percentage of survival of the non-cold-acclimated diabetic rats (Expt.l) was 19% compared with 79% of the diabetic cold-acclimated animals (Expt. 2). There were no deaths in the control groups. 5. 5. Cold-acclimated diabetic rats maintained a near-normal thermogenic response after noradrenaline injection. This response was impaired in non-cold-acclimated diabetic rats. 6. 6. The results of these experiments suggest that the enhanced cold-tolerance of diabetic cold-acclimated rats could be related to the increased sympathetic activity and enhanced insulin sensitivity in thermogenic tissues, such as brown fat. © 1987.

Müller cells and diabetic retinopathy.

Müller cells provide nutrition for neural cells. We studied the structure and ultrastructure of Müller cells in the retina of thirty 3-month old Wistar rats, divided equally into 3 groups: normal rats, alloxan diabetic rats and treated alloxan diabetic rats, 1 and 12 months after induction of diabetes. We observed that the Müller cell nuclei under light microscope examination had hexagonal shape and higher density than the other nuclei. Differences between groups could be observed only by electron microscopy. In the diabetic rats, Müller cells presented dispersion of nuclear chromatin and electrondense nuclear granulations, with the presence of increased glycogen, dense bodies and lysosomes in the cytoplasm. The alterations were more frequent in the perivascular region and at 12 months. The treated diabetic rats exhibited some alterations we observed in diabetic rats, but these alterations were less intense. We conclude that, despite the treatment, the diabetic retinopathy continues to evolve.

Role of different proteolytic pathways in degradation of muscle protein from streptozotocin-diabetic rats

In vitro rates of overall proteolysis and the activities of four different proteolytic pathways (lysosomal, Ca2+ dependent, ATP dependent, and ATP independent), as well as rates of protein synthesis, were measured in soleus and extensor digitorum longus (EDL) muscles from streptozotocin- diabetic rats. In the acute phase (1-3 days) of diabetes, there was an increase in overall proteolysis that coincided with an increased activity of the Ca2+-dependent pathway in both soleus and EDL and of the ATP-dependent pathway in EDL. After longer periods (5-10 days) of diabetes, the overall rate of protein degradation decreased and reached values similar to or even lower than those of controls as a result of a reduction in the activities of Ca2+-dependent and ATP-dependent pathways. No change was detected at any time interval in the activity of the intralysosomal proteolytic system in muscles from diabetic animals. Rates of protein synthesis were already reduced 24 h after diabetes induction and decreased further thereafter. Insulin treatment restored to normal the activities of the proteolytic pathways and rates of protein synthesis.

The hemolytic uremic syndrome as a complication of adriamycin nephropathy

Rats treated with two injections of adriamycin (week 0 and week 12) developed glomerusclerosis and severe tubulointerstitial lesions as described in the literature. In addition, a number of glomerular alterations were present. These included capillary loop dilation, insudation of eosinophilic material, necrosis, duplication of the glomerular basement membrane, severe mesangiolysis with disruption of the mesangial matrix and segmental double- contours. The renal arterioles and interlobular arteries showed endothelial cell swelling. The subendothelial space was infiltrated by fibrinoid material and there was intensive fibrinoid necrosis of the wall of both arteries and arterioles extending into the glomerular tuft. These alterations were very similar to those observed in the hemolytic uremic syndrome. This observation suggests that the two injections of adriamycin, with a long interval in between them, might induce renal lesions similar to those observed in the hemolytic uremic syndrome.

Effects of cyclosporine on adriamycin induced nephropathy

The effect of cyclosporine on adriamycin nephropathy was studied over both short (6 weeks) and long (26 weeks) periods. In the short-term study, cyclosporine was introduced 2 weeks after nephritis induction and in the long-term study, 14 weeks after the first adriamycin injection. The animals with adriamycin nephropathy treated with cyclosporine, studied for 6 weeks, developed proteinuria with increased renal size and glomerular area. The nephrotic animals treated with cyclosporine showed less proteinuria than the nephrotic control animals. There were no differences in glomerular area, creatinine clearance or serum creatinine and kidney weight between the treated nephrotic group and the health control group. The nephrotic animals, studied for the longer period, developed intense proteinuria, decreased creatinine clearance, glomerular necrosis and sclerosis, severe tubulointerstitial nephritis, increased hydroxyproline concentration and tubulointerstitial area. No difference was observed between the nephrotic animals treated with cyclosporine and those not treated. In conclusion, Cyclosporine A reduced proteinuria, glomerular hypertrophy and kidney weight in rats with short-term nephropathy but had no effect on the established nephropathy.

Effect of allopurinol in the course of adriamycin induced nephropathy

The role of superoxide in adriamycin-induced nephropathy (single dose; i.v. 3 mg/kg) has been studied by blocking superoxide synthesis through the administration of allopurinol (500 mg/L in drinking water). In Experiment I (EI), allopurinol administration was started 3 days prior to nephropathy induction and continued until day 14. In Experiment II (EII) allopurinol administration was started 2 weeks after nephropathy induction and was maintained until the end of the experiment (26 weeks). Affected glomeruli frequency and tubulointerstitial lesion index (TILI) were determined at Weeks 2 and 4 (EI) and Week 26 (EII). In EI, and 24 h mean proteinuria in the nephrotic control group (NCG-I) differed from that of the treated nephrotic group (TNG-I) at Week 1 (TNG = 33.3 ± 6.39 mg/24 h; NCG = 59.8 ± 6.3 mg/24 h; p < 0.05) and 2 (NCG-I = 80.0 ± 17.5 mg/24h; TNG-I = 49.1 ± 8.4 mg/24 h; p < 0.05). No glomerular alterations were observed and TILI medians were not different in both nephrotic groups at week 2 (NCG-I = 1+: TNG = 1+) and 4 (NCG = 4+; TNG = 4+). In EII, NCG-II and TNG-II presented different 24 h proteinuria values only at Week 6, (136.91 ± 22.23 mg/24 h ad 72.66 ± 10.72 mg/24 h, respectively; p < 0.05). Between nephrotic groups, there was no statistical difference in the median of affected glomeruli (CNG-II = 56%; TNG-II = 48% and TILI (NCG-II = 8+; TNG-II = 9+). Thus, allopurinol was associated with a transient reduction in proteinuria and it did not alter the progression of the nephropathy.

Role of ticlopidine on adriamycin-induced nephropathy

The effect of ticlopidine on rats with adriamycin nephropathy was observed during 26 weeks. In the ticlopidine-treated nephrotic animals (TNG), proteinuria was less than in the untreated nephrotic animals (NG), but this difference was significant only at week 6 (TNG = 47.27 ± 16.52 versus NG = 100.08 ± 13.83 mg/24h, p < 0.01) and week 26 (TNG = 157.00 ± 28.73 versus NG = 217.00 ± 21.73 mg/24h, p< 0.01) after ADR injection. NG presented severe tubulointerstitial abnormalities with a tubulointerstitial lesion index of 3+. No difference in glomerular lesions was observed among the groups (NG median = 6%, TNG median = 4% and TCG median = 2%). The tubulointerstitial lesion index of TNG was less intense (median = 2+) but not different from those of the control groups (CG median = 1+; TCG median = 0+) nor NG (median = 3+). We concluded that the treatment with ticlopidine produced some partially beneficial effects but did not prevent the development of adriamycin-induced nephropathy.

Importance of early and continuous use of protein restriction on the progression of adriamycin nephropathy

Three experimental protocols were carried out with the aim of evaluating the role of protein restriction on the progression of the established adriamycin-induced nephropathy, and whether the protective effect of the diet persists after the diet is discontinued. The effect of a low protein diet (LPD) was studied for 6 weeks in protocol 1, 16 weeks in protocol 2 and for 28 weeks in protocol 3. In protocol 3, one group (LL) received LPD and another (NN) was given a normal protein diet (NPD). A third group (LN) received LPD for 16 weeks and then NPD for 12 weeks and a fourth group (NL) was fed NPD for 16 weeks and then LPD for 12 weeks. In protocol I the tubulo- interstitial index (TILl) of rats on LPD (Md = 2, P25 = 0.0; P75 = 3.5) after six weeks, was smaller than that of the animals on NPD (Md = 6.0; P25 = 3.0; P75 = 8.0; p < 0.05). In protocol 2, the group taking LPD presented an area of interstitial fibrosis (IF) (Md= 0.5%, P25 0.2%; P75 = 1.9%) smaller than that of the NPD group (Md = 6.8%; P25 = 5.2%; P75 = 7.1%; P < 0.05). No significant difference in the area of glomerulosclerosis (GSA) was observed between the animals on LPD (Md = 0.0%; P25 = 0.0%, P75 = 0.0%) and NPD (Md = 0.37%; P25 = 02% P75 = 1.25%; p > 0.05). In protocol 3, the group LL showed GSA (Md = 1.3%; P25 0.6%, P75 = 2.5%) and IF (Md = 3.60/0; P25 = 1.6%; P75 = 5.9%) smaller that those of LN (GSA Md = 10.1%; P25 = 6.6%; P75 = 14.8%; IF; Md = 17.3%; P25 = 14.1%; P75 = 24,5%), NL (GSA: Md = 9.1%; P25 = 5,8%; P75 = 11.7%; IF; Md = 25.0%; P25 = 20.4%; P75 = 30%), and NN (GSA: Md = 6. 75%; P25 = 4.9%; P75 = 11.7%; IF: Md = 20.9%; P25 = 16.2%; P75 = 32.4%). In conclusion, in order to be effective, LPD must be introduced early and maintained for a long period of tune.

Evaluation of toxicity after one-months treatment with Bauhinia forficata decoction in streptozotocin-induced diabetic rats

Background: Previous experiments have shown that a decoction of Bauhinia forficata leaves reduces the changes in carbohydrate and protein metabolism that occur in rats with streptozotocin-induced diabetes. In the present investigation, the serum activities of enzymes known to be reliable toxicity markers were monitored in normal and streptozotocin-diabetic rats to discover whether the use of B. forficata decoction has toxic effects on liver, muscle or pancreas tissue or on renal microcirculation. Methods: An experimental group of normal and streptozotocin-diabetic rats received an aqueous decoction of fresh B. forficata leaves (150 g/L) by mouth for 33 days while a control group of normal and diabetic rats received water for the same length of time. The serum activity of the toxicity markers lactate dehydrogenase, creatine kinase, amylase, angiotensin-converting enzyme and bilirubin were assayed before receiving B. forficata decoction and on day 19 and 33 of treatment. Results: The toxicity markers in normal and diabetic rats were not altered by the diabetes itself nor by treatment with decoction. Whether or not they received B. forficata decoction the normal rats showed a significant increase in serum amylase activity during the experimental period while there was a tendency for the diabetic rats, both treated and untreated with decoction, to have lower serum amylase activities than the normal rats. Conclusions: Administration of an aqueous decoction of B. forficata is a potential treatment for diabetes and does not produce toxic effects measurable with the enzyme markers used in our study. © 2004 Pepato et al; licensee BioMed Central Ltd.

Choroidal vasculature in diabetic rats

This study aims to evaluate the diabetic influence on the choroidal vessels morphology. Twenty Wistar rats were divided into a control (CG) and a diabetic group (DG). The animals had the diabetes induced by an intra-venous injection of Alloxan (42 mg/kg). Transmission electron microscopy analysis focusing the choroidal vessels was done one (T2) and twelve (T3) months after the diabetes induction. The CG rats in T3 showed vesicles and dense bodies in the endothelial and pericytic cells; the same structures were observed in the DG at T2. The DG rats in T3 had even more and intense changes than the T2DG rats. The morphological evaluation indicates that the choroidal vessels are affected in diabetes and the disease accelerates degenerative processes in the rat choroidal vasculature.

The role of myofibroblasts and interstitial fibrosis in the progression of membranous nephropathy

Renal interstitial fibrosis has been observed in a large number of nephropathies and contributes to the progressive deterioration of renal function. Myofibroblasts have been implicated in the reparative process of tissue injury, including renal scarring secondary to glomerular diseases. We performed a retrospective study on 28 patients with biopsy-proven primary membranous nephropathy, to determine whether interstitial myofibroblasts and tubulointerstitial lesions correlated with renal function at follow-up. Tubulointerstitial pathology was evaluated by morphometric and semiquantitative methods. Interstitial myofibroblasts were counted; 24-hour urinary protein and serum creatinine at the time of diagnosis and at the end of follow-up were available for all the patients. There were 20 males and 8 females, age 2-67 years (mean 42.3±153), most of them with nephrotic syndrome (78.6%). The final renal function had deteriorated in 16 patients (57.1%) and in 5 patients (17.8%) reached end-stage. The renal outcome was correlated with histological changes. We found a positive correlation between the severity of tubulointerstitial damage and the deterioration of the final serum creatinine (r 2=0.185; p=0.016). Myofibroblasts did not predict impaired renal function at the final follow-up. The current data do not support previous suggestions that myofibroblasts are a useful a predictor of end-stage renal disease.

The effect of low intensity laser therapy on wound healing in Streptozotocin-induced diabetic rats

Diabetes Mellitus is a condition that results in a delay of the wound healing process, that is associated with an insufficient production of collagen, a decrease of the amount of collagen fibrils and deficient blood flow in the wound area. It is suggested that Low Intensity Laser Therapy acts by improving wound healing in normal organisms, accelerating tissue regeneration. The aim of this work was to investigate the biostimulatory effect of the HeNe laser irradiation, at 632.8 nm, on wound healing in 15 male rats suffering from diabetes induced by Streptozotocin, compared to 15 control diabetic animals. Irradiation parameters were: laser power of 15mW, exposition time of 17 s., irradiated area of 0.025 cm 2 and laser energy density of 10 J/cm 2. Full-thickness skin squared samples, with 5 mm of non-injured tissue around the wound, were obtained at 4, 7 and 15 days after wounding procedure (5 treated and 5 control animals each time). The histopathologic analysis performed by haematoxylin-eosin staining. Results suggested that the irradiation of diabetic rats was efficient for wound healing. Treated group presented better quality of the wound tissues by the macroscopic observation than control group and the microscopic analysis demonstrated that treated animals had better histopathologic evaluation than non treated.

Porous polyethylene for tissue engineering applications in diabetic rats treated with calcitonin: Histomorphometric analysis

Purpose: The purpose of this work was to study the bone tissue reaction after porous polyethylene (Polipore) implantation into surgical defects in the parietal bones of rats with streptozotocin-induced diabetes, treated with salmon calcitonin. Materials and Methods: Porous polyethylene implants were placed in bone defects created in 36 adult female rats. The rats were divided into 3 equal groups: diabetic treated with calcitonin (DCa), diabetic (D), and control (C). The animals of the DCa group received applications of salmon calcitonin on alternating days immediately after the surgery until sacrifice. The rats were sacrificed after 15, 30, 60, and 90 days, and the defects were examined histologically and statistically through histomorphometric analysis. Results: Histomorphometric analysis showed that there was no statistically significant difference in the mean quantity of inflammatory cells among all study groups after 15 and 90 days. At 30 days, a statistically significant difference was observed between the D and C groups and the D and DCa groups. At 60 days, there was no statistically significant difference between the D and DCa groups. Discussion: Porous polyethylene can be considered an option for implant material when there are investigations that prove its biocompatibility and stability in the host tissues. Salmon calcitonin positively aided the bone repair and attenuated the inflammatory response until 30 days after the surgery. Conclusion: Porous polyethylene was tolerated by the host tissues in all groups, and moderate chronic inflammatory reaction was observed up to the 90-day period. Salmon calcitonin attenuated the inflammatory response up until 30 days.

Morphometric and quantitative evaluation of the NADH-diaphorase positive myenteric neurons of the jejunum of streptozotocin-diabetic rats supplemented with acetyl-L-carnitine

In this study we investigated the effect of the acetyl-L-carnitine (ALC) supplementation on the myenteric neurons of the jejunum of rats made diabetic at the age of 105 days by streptozotocin (35 mg/kg body weight). Four groups were used: non-diabetic (C), non-diabetic supplemented with ALC (CC), diabetic (D), diabetic supplemented with ALC (DC). After 15 weeks of diabetes induction the blood was collected by cardiac puncture to evaluate glycaemia and glycated haemoglobin. Next the animals were killed and the jejunum was collected and subjected to whole-mount preparation to evidence the myenteric neurons through the histochemical technique of the NADH-diaphorase. The neuronal counts were made in 80 microscopic fields, in tissue samples of five animals of each group. The profiles of the cell bodies of 1000 neurons per group were analysed. Diabetes induced a significant increase in the area of the cell body and decrease in the number of NADH-diaphorase positive myoenteric neurons. ALC suplementation to the diabetic group promoted smaller hypertrophic effects and less neuronal loss than in the myoenteric neurons of the diabetic rats, and in addition diminished the body weight decrease and reduced the fasting glycaemia. © 2005 Blackwell Verlag.

Microscopic, morphometric and ultrastructural analysis of anastomotic healing in the intestine of normal and diabetic rats

The purpose of this study was to investigate if experimental alloxanic diabetes could cause qualitative changes in intestinal anastomoses of the terminal ileum and distal colon in rats, as compared to controls. 192 male Wistar rats, weighing ± 300g were split into four experimental groups of 48 animals each, after 3 months of follow-up: a control group with ileum anastomoses (G1), a control group with colon anastomoses (G2), a diabetic group with ileum anastomoses (G3) and a diabetic group with colon anastomoses (G4). Animals were evaluated and sacrificed on days 4, 14, 21 and 30 after surgery, and fragments of the small and large intestine where the anastomoses were performed were removed. Samples from 6 animals from each sacrifice moment were submitted to ultrastructural analysis of the collagen fibers using a scanning electron microscope and samples from another 6 animals were submitted to histopathology and optical microscopy studies using picrosirius red-staining. Histopathological analysis of picrosirius red-stained anastomosis slides using an optical microscope at 40x magnification showed that the distribution of collagen fibers was disarranged and also revealed a delay in scar tissue retraction. The morphometric study revealed differences in the collagen filled area for the ileum anastomoses 14 days post surgery whereas, in the case of colon anastomoses, differences were observed at days 4 and 30 post surgery, with higher values in the diabetic animals. Ultrastructure analysis of the ileum and colon anastomoses using a scanning electron microscope revealed fewer wide collagen fibers, the presence of narrower fibers and a disarranged distribution of the collagen fibers. We conclude that diabetes caused qualitative changes in scar tissue as well as in the structural arrangement of collagen fibers, what could explain the reduced wound strength in the anastomosis of diabetic animals. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart.