Neonatally Induced Mild Diabetes in Rats and Its Effect on Maternal, Placental, and Fetal Parameters

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Neonatally-induced diabetes: lipid profile outcomes and oxidative stress status in adult rats

INTRODUÇÃO: Modelos experimentais são desenvolvidos com propósito de ampliar o entendimento dos mecanismos fisiopatológicos envolvidos no diabete. Os achados experimentais levam ao desenvolvimento de tratamentos alternativos para a manutenção das condições metabólicas normais. Existem vários estudos sobre o diabete induzido por streptozotocin mimetizando o quadro clínico do DM2. No entanto, a interação entre os níveis de glicose, perfil lipídico e estresse oxidativo nestes animais são escassos. Portanto, o objetivo do trabalho foi avaliar estes parâmetros em ratas Wistar adultas com diabete induzido com streptozotocin no período neonatal. MÉTODOS: Fêmeas recém-nascidas receberam streptozotocin (70mg/Kg, ip) no 5º dia de vida (n5-STZ). A glicemia foi medida no terceiro e quarto meses de vida dos animais. No final do quarto mês de vida, amostras de sangue foram coletadas e processadas para a dosagem de lipídios e marcadores de estresse oxidativo. RESULTADOS: A glicemia das ratas do grupo n5-STZ foi significativamente maior comparada às ratas do grupo controle (p<0,05). Não houve alteração nos níveis de colesterol total e triglicérides, peroxidação lipídica (TBARS), atividade da SOD e determinação da GSH-t (p>0,05) nas ratas n5-STZ em relação às ratas do grupo controle. No entanto, houve diminuição significativa no HDL-colesterol (p<0,05). CONCLUSÃO: Este modelo de indução de diabete em ratas causou hiperglicemia (120-360mg/dL), caracterizando o diabete moderado. Essa glicemia levou a alterações no HDL-colesterol, a qual não foi suficiente para prejudicar a atividade das enzimas antioxidantes ou marcadores da peroxidação lipídica na vida adulta. Além disso, esta investigação experimental contribuiu para entender os diferentes resultados encontrados em outros modelos deindução do diabete moderado em animais de laboratório, como também para a melhor compreensão dos mecanismos fisiopatológicos do diabete moderado ou da hiperglicemia em humanos.

Infiltrating CD8(+) T lymphocytes, natural killer cells, and expression of IL-10 and TGF-beta 1 in chemically induced neoplasms in male Wistar rats

The present study aimed to estimate the number of CD8(+) T and natural killer (NK) infiltrating cells and the expression of interleukin-10 (IL-10) and transforming growth factor beta 1 (TGF-beta1) in chemically induced neoplasms in an initiation-promotion bioassay for carcinogenesis. Male Wistar rats were treated with N-nitrosodiethylamine, N-methyl-N-nitrosourea, N-butyl-N-(4-hydroxybutyl) nitrosamine, dihydroxy-di-N-propylnitrosamine, and 1,2-dimethylhydrazine for 4 weeks. Two groups were subsequently exposed through diet to phenobarbital (0.05%) or 2-acetylaminofluorene (0.01%) for 25 weeks. An untreated group was used as a control. Immune cells and cytokines were immunohistochemically evaluated in neoplasms and in surrounding normal tissues at the liver, kidneys, lung, and small and large intestines. When compared to the respective normal tissues, an increased number of NK cells was verified infiltrating the colon, lung, and kidney neoplasms, while the number of CD8+ T cells decreased in the intestine and lung neoplasms. Expression of IL-10 was found mainly in kidney tumors. TGF-beta1 was expressed mainly in the liver and kidneys tumors. The results indicate that the differential occurrence of immune cells between neoplastic and normal tissues could be dependent upon tumor microenvironment.

Progression of nephropathy after islet of langerhans transplantation in alloxan-induced diabetic rats

We studied the effects of islet of Langerhans transplantation (IT) on the kidney lesions of rats with alloxan-induced diabetes. Forty-five inbred male Lewis rats were randomly assigned to 3 experimental groups: group Gl included 15 non-diabetic control rats (NC), group GIT included 15 alloxan-induced diabetic rats (DC), and group III included 15 alloxan-induced diabetic rats that received pancreatic islet transplantation prepared by nonenzymatic method from normal donor Lewis rats and injected into the portal vein (IT). Each group was further divided into 3 subgroups of 5 rats which were sacrificed at 1, 3, and 6 months of follow-up, respectively. Clinical and laboratorial parameters were recorded in the mentioned periods in the 3 experimental groups. For histology, the kidneys of all rats of each subgroup were studied and 50 glomeruli and 50 tubules of each kidney were analyzed using light microscopy by two different investigators in a double blind study. The results showed progressive glomerular basement membrane thickening (GBMT), mesangial enlargement (ME), and Bowman's capsule thickening (BCT) in the 3 experimental groups throughout the follow-up. These alterations were significantly more severe in DC rats at 6 months when compared to NC rats (p < 0.01). However, the degree of GBMT, ME, and BCT observed in DC rats was not statistically different from IT rats at 1, 3, and 6 months. In addition, Armanni-Ebstein lesions of the tubules (AE) and tubular lumen protein (PRO) observed in DC rats were also observed in IT rats all over the study. These lesions were never present in NC rats. We conclude that IT did not prevent progression of kidney lesions in alloxan-induced diabetic rats within 6 months after transplantation.

Aberrant crypt foci and colon cancer: comparison between a short- and medium-term bioassay for colon carcinogenesis using dimethylhydrazine in Wistar rats

Aberrant crypt foci (ACF) in the colon of carcinogen-treated rodents are considered to be the earliest hallmark of colon carcinogenesis. In the present study the relationship between a short-term (4 weeks) and medium-term (30 weeks) assay was assessed in a model of colon carcinogenesis induced by dimethylhydrazine (DMH) in the rat. Six-week-old male Wistar rats were given subcutaneous injections of DMH (40 mg/kg) twice a week for 2 weeks and killed at the end of the 4th or 30th week. ACF were scored for number, distribution pattern along the colon and crypt multiplicity in 0.1% methylene-blue whole-mount preparations. ACF were distinguished from normal crypts by their larger size and elliptical shape. The incidence, distribution and morphology of colon tumors were recorded. The majority of ACF were present in the middle and distal colon of DMH-treated rats and their number increased with time. By the 4th week, 91.5% ACF were composed of one or two crypts and 8.5% had three or more crypts, while by the 30th week 46.9% ACF had three or more crypts. Thus, a progression of ACF consisting of multiple crypts was observed from the 4th to the 30th week. Nine well-differentiated adenocarcinomas were found in 10 rats by the 30th week. Seven tumors were located in the distal colon and two in the middle colon. No tumor was found in the proximal colon. The present data indicate that induction of ACF by DMH in the short-term (4 weeks) assay was correlated with development of well-differentiated adenocarcinomas in the medium-term (30 weeks) assay.

Long-term effects of insulin therapy, islet transplantation, and pancreas transplantation in the prevention of glomerular changes in kidneys of alloxan-induced diabetic rats

Groups of inbred alloxan-induced diabetic rats were treated with insulin (I), islets (IT), or pancreas transplantation (PT). Nondiabetic (N) and untreated diabetic (D) control groups were concurrently included. Each group was divided into five subgroups of 10 rats and killed after follow-up of 1, 3, 6, 9, and 12 months. Clinical and laboratory parameters were recorded, and kidney ultrastructural and morphometric analyses performed in each 12-month subgroup, namely glomerular basement membrane (GM) thickening, podocyte number, and number/extension of slit diaphragms (S). Rats from the I group showed poor metabolic control of diabetes compared with N group control rats. However, successfully transplanted rats (IT and PT) had complete restoration to normal levels for all metabolic parameters. GM thickening was significantly higher in diabetic compared with control rats. In contrast, the numbers of podocytes and slits as well as slit extensions were significantly decreased. Insulin therapy did not prevent any alterations upon comparison of diabetic vs control rats. Despite good metabolic control in IT rats, the degree of kidney lesion control never compared with that achieved in PT rats. In this group all glomerular changes were similar to the age-dependent lesions observed in control rats. We conclude that either IT or PT may be a good option for diabetes treatment, although pancreas transplantation seems to be the most effective treatment to control chronic complications.

Effects of uterine cervix constriction on Wistar rats

Purpose: To verify if uterine cerclage can induce craniosynostosis or any cranial deformity in new born Wistar rats. Methods: One pregnant female Wistar rat underwent laparotomy on day 18 of gestation and the uterus cervix was closed with a 3-0 nylon suture to avoid delivery, that occurs normally on the 21 day. The suture was released after 48 hours beyond the normal gestation period. The female rat delivered 11 pups. Six surviving rats from the delivery (group A - constrained group). Two rats were born from another mother and in the same age were used as control group (group B - 2 nonconstrained controls) were allowed to grow. They were sacrificed 1.2 years after their birth all the eight animals. Linear measurement, routine histology and computed tomography of the skull were performed at the time of their death to evaluate the cranial asymmetries by mesurements of the anatomical landmarks of the craniofacial skeleton of the rats on the two groups and compared then. Results: We did not observe statistically significant differences in any of the compared measurements (p>0.05) obtained through the morphologic and radiologic methods. Histologic examinations did not reveal any sign of premature fusion or suture imbrications. Critical decrease in longitudinal body size was noticed as the limbs too in all the animals of group A. Conclusion: Constriction of uterine cervix leads to fetus suffering, even death for a few animals, associated to small body size, but not to craniosynostosis.

Osteocalcin immunolabeling during the alveolar healing process in ovariectomized rats treated with estrogen or raloxifene

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Histomorphometric evaluation of periodontal compression and tension sides during orthodontic tooth movement in rats

OBJECTIVE: The purpose of this study was to evaluate the thickness of the periodontal ligament of rat molars during orthodontic tooth movement (OTM). METHODS: Thirty Wistar rats were divided into three groups of 10 animals each: GI, GII and GIII and the mice were euthanized at 7, 14 and 21 days, respectively. Experimental subjects were compared to their respective controls by the Mann-Whitney test. Comparison of values between compression and tension sides were performed during the same and different time periods through Analysis of Variance (ANOVA), Kruskal-Wallis test and, subsequently, Tukey's test. RESULTS: Groups GI and GII showed decreased PDL size in the apical regions of the mesiobuccal root and in the cervical region of the distobuccal root. There was also an increased PDL in the cervical regions of the mesiobuccal root, apical region of the distobuccal root and middle region of both roots. CONCLUSION: The reduction and increase in PDL size were seen in the same root, which characterizes tooth inclination. The apical, middle and cervical regions were compared with one another in each time period and at three times: 7, 14 and 21 days. They were also compared in each region, confirming a tipping movement in GI and GII and a gradual decreased intensity between GI to GII, reaching normal dimension in GIII.

Effect of low-level laser therapy on bone repair: Histological study in rats

Background and Objectives: Bone remodeling is characterized as a cyclic and lengthy process. It is currently accepted that not only this dynamics is triggered by a biological process, but also biochemical, electrical, and mechanical stimuli are key factors for the maintenance of bone tissue. The hypothesis that low-level laser therapy (LLLT) may favor bone repair has been suggested. The purpose of this study was to evaluate the bone repair in defects created in rat lower jaws after stimulation with infrared LLLT directly on the injured tissue.Study Design/Materials and Methods: Bone defects were prepared on the mandibles of 30 Holtzman rats allocated in two groups (n = 15), which were divided in three evaluation period (15, 45, and 60 days), with five animals each. control group-no treatment of the defect; laser group-single laser irradiation with a GaAlAs semiconductor diode laser device (lambda = 780 nm; P = 35 mW t = 40 s; circle minus = 1.0 mm; D = 178 J/cm(2); E = 1.4 J) directly on the defect area. The rats were sacrificed at the preestablished periods and the mandibles were removed and processed for staining with hematoxylin and eosin, Masson's Trichrome and picrosirius techniques.Results: the histological results showed bone formation in both groups. However, the laser group exhibited an advanced tissue response compared to the control group, abbreviating the initial inflammatory reaction and promoting rapid new bone matrix formation at 15 and 45 days (P < 0. 05). on the other hand, there were no significant differences between the groups at 60 days.Conclusion: the use of infrared LLLT directly to the injured tissue showed a biostimulating effect on bone remodeling by stimulating the modulation of the initial inflammatory response and anticipating the resolution to normal conditions at the earlier periods. However, there were no differences between the groups at 60 days.

Effect of diabetes mellitus and insulin therapy on bone density around osseointegrated dental implants: a digital subtraction radiography study in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Lesions of the commissural nucleus of the solitary tract reduce arterial pressure in spontaneously hypertensive rats

It has been suggested that increased sympathetic activity and arterial chemoreceptors are important for the high blood pressure in spontaneously hypertensive rats (SHR). Electrolytic lesions of the commissural nucleus of the solitary tract (commNTS) abolish (1) the cardiovascular responses to chemoreflex activation with potassium cyanide (KCN) in normotensive rats and (2) the hypertension that follows acute aortic baroreceptor denervation in rats. Therefore, in this study we investigated the effects of electrolytic lesions of the commNTS on basal mean arterial pressure (MAP), baroreflex, and chemoreflex in SHR and in normotensive control Wistar-Kyoto (WKY) and Wistar rats. CommNTS lesions elicited a dramatic fall in MAP to normal levels during the period of Study (from the first to fourth day following lesions) in SHR and almost no changes in WKY and Wistar rats. The pressor responses to chemoreflex activation with KCN tested in the days 1 and 4 after commNTS lesions were abolished in SHR and in normotensive strains. The reflex tachycardia induced by sodium nitroprusside was also attenuated in days 1 and 4 after commNTS lesions in SHR, WKY, and Wistar rats. The data suggest that the integrity of commNTS is important for the maintenance or high blood pressure in SHR and for the reflex responses dependent on sympathetic activation either in SHR or in normotensive strains.

Short-term induction of thrombocytopenia delays periodontal healing in rats with periodontal disease: participation of endostatin and vascular endothelial growth factor

Background and Objective:Platelets contain factors, including VEGF and endostatin, that can modulate the healing process. We evaluated the effects of severe thrombocytopenia on periodontal healing in rats and determined the contribution of VEGF and endostatin to the healing process.Material and Methods:Rats were distributed into three test groups and two control groups. Cotton ligatures were placed at the gingival margin level of the lower first molar in the test groups. Sham-operated rats and rats in one of the periodontitis groups were killed 15 days later. Rats in the remaining two periodontitis groups had the ligatures removed in order to study the spontaneous recovery from the periodontal disease 15 days later, and these rats were treated with rabbit antiplatelet serum, in order to induce thrombocytopenia, or normal rabbit serum. An additional group without ligatures received antiplatet serum in the same period.Results:After ligature removal, rats treated with normal rabbit serum showed reduced myeloperoxidase activity, decreased alveolar bone loss and increased numbers of blood vessels. Thrombocytopenia caused a delay in alveolar bone regeneration, a decrease in the number of vessels and a modest decrease in myeloperoxidase activity. In the rats with periodontitis, serum endostatin concentrations were slightly decreased and serum VEGF remained unchanged compared with sham-operated animals. After ligature removal, a significant VEGF increase and endostatin decrease were observed in the rats treated with normal rabbit serum. Thrombocytopenia led to a dramatic fall in both VEGF and endostatin concentrations.Conclusion:Thrombocytopenia leads to a delay of periodontal healing in the situation of experimental periodontitis, which might be mediated in part by a decrease in the serum concentration of VEGF and endostatin derived from the platelets. However, other factors derived from the platelets may also have contributed to a delay of periodontal healing in the rats with thrombocytopenia.

Epididymis-specific pathologic disorders in rats exposed to gossypol from weaning through puberty

Previous work in our laboratory revealed that the pubertal period of reproductive development in the male rat was particularly vulnerable to gossypol exposure, with a higher frequency of round structures in the lumen of the cauda epididymidis in the treated rats. Herein, we utilized hemicastration and electron microscopy to confirm that the epididymis is a definitive target of gossypol. Although exposure to gossypol from weaning through puberty caused a significant decrease in daily sperm production, as well as in the concentration of sperm in the epididymis, serum testosterone levels and reproductive organ weights were not altered. In gossypol treated rats, sperm morphology was compromised severely, but the epithelium in testis and epididymis appeared morphologically normal. Ultrastructural examination revealed that round structures, present only in gossypol exposed males, represented: (1) principal cells exfoliated from the epididymal epithelium; (2) epididymal epithelial cell cytoplasm containing degenerating sperm; and (3) degenerating epithelial cells, consisting of vesicles and particles of different sizes, forms and densities. Taken together, the data confirm that gossypol targets the epididymis, disturbing both the structure and function of this organ, and presumably disrupts sperm maturation.

The importance of androgen on noradrenergic responses of vas deferens isolated from acutely stressed rats

This study investigated the importance of androgen on responses to alpha and beta (norepinephrine) and alpha(1) (phenylephrine and methoxamine) agonists in vasa deferentia isolated from adult, immature, cryptorchid, and castrated rats submitted to swimming-induced acute stress. The participation of adrenergic nervous terminals was also investigated. Acute stress was shown to induce a significant subsensitivity to norepinephrine only in vas deferens from adult rats with normal levels of androgens. In addition, sympathetic denervation of the vas deferens prevented the appearance of subsensitivity. Subsensitivity was not seen when the experiments were carried out using phenylephrine and methoxamine. This shows that subsensitivity to norepinephrine in this acute stress situation may depend on other factors such as neuronal uptake, but not on alpha(1)-adrenoceptor response. Thus, when animals are exposed to acute stressogenic situations, this subsensitivity requires physiological levels of androgens to establish, and may also be involved in body homeostasis. (C) 1999 Academic Press.