João Carlos Domingues Repka

Avaliação espectrofotométrica do azul de Evans na reação inflamatória da córnea: estudo experimental em coelhos

PURPOSE: To detect Evans blue in the normal cornea, attempting to determine the period of maximum concentration of the dye after intravenous injection and to study vascular permeability in an animal model of corneal inflammation induced by alkali burn, after Evans blue injection. METHODS: Fifty rabbits were divided into 3 groups. Group I (25 animals): 20 mg/kg Evans blue were injected and the animals were sacrificed after 8, 10, 12, 14 and 16 hours. The corneas were removed and the dye concentration was measured by a spectrophotometric micromethod. Group II: in 15 animals, 10 hours after injection, the corneas were centrally fragmented with number 6, 8 and 10 mm trephines. Evans blue was extracted the same way as in Group I. Group III: alkali burn was induced in the cornea of the right eyes of 10 animals, using 1 N NaOH. Five days after the procedure, the animals were sacrificed. Ten hours prior to sacrifice, Evans blue was injected. The left corneas were the control. RESULTS: In group I, mean Evans blue concentration is at 10 h: 15.28 ± 0.09 mg/mg. In group II, mean Evans blue concentrations are: 6 mm: 0.93 ± 0.01mg/mg; 8 mm: 1.20 ± 0.06 mg/mg; 10 mm: 1.32 ± 0.05 mg/mg. In group III, mean Evans blue concentrations are: right eyes (alkali burns): 23.74 ± 2.64 mg/mg and left eyes (control): 16.71 ± 2.04 mg/mg. CONCLUSIONS: This is the first time that Evans blue has been quantified in rabbits cornea. It was possible to detect the dye from 8 to 16 hours after intravenous injection. We can conclude that Evans blue detection is a good method to quantify the alteration of vascular permeability in rabbits cornea.

A influência da nicotina na densidade de colágeno em cicatrizes cutâneas, em ratos

OBJECTIVE To study the inflammatory reaction and deposition of collagen in the healing of cutaneous injuries under the influence of nicotine. METHODS The scars of abdominal injuries in rats were analyzed, which had been treated with nicotine, 2 mg/kg/d, and compared with those of control rats. Treatment was begun seven days prior to operation and was maintained for seven or fourteen days after surgery. The histological cuts were stained with hematoxylin and eosin and through the established scores the intensity and type of inflammatory reaction was identified. Histological cuts stained by Sirius Supra red F3BA enabled the identification of the collagen density. RESULTS There was no difference in intensity of the inflammatory reaction after seven days (p=0.165), nor after fourteen days (p=0.684). There was no significant difference in the type I collagen density in the evaluation carried out after seven days (p=0.912) and after fourteen days (p=0,211). The control group had more type III collagen after seven days (p=0.004), but after fourteen days there was no significant difference (p=0.720). Although the total quantification of collagen was higher in the control group, there was no significant difference at any time during this study (p=0.103 after seven days and p=0.549 after fourteen days). CONCLUSION In the scars of the animals treated with nicotine in comparison with the control group, there was no difference in the intensity of the inflammatory process, nor in collagen density.

Ação do tacrolimus na pancreatite aguda experimental induzida pela arginina

OBJECTIVE To determine whether tacrolimus administered to rats, in the presence of pancreatitis induced by L-Arginine, interferes with the serum levels of amylase and glucose and the histological pattern of the pancreatic parenchyma. METHODS Forty Wistar rats were divided into four groups with 10 rats each: control group (C), tacrolimus group (T), pancreatitis group (P) and pancreatitis-tacrolimus group (PT). We evaluated serum levels of amylase, glucose, and tacrolimus and made histological assessments of the pancreas. Induction of pancreatitis was made by inoculation of L-Arginine at a dose of 500 mg/100g body weight intraperitoneally, and tacrolimus treatment at a dose of 1ìg/kg subcutaneously for four days. RESULTS Serum amylase was higher (p = 0.0000) in groups PT, P and T than in the control group. The PT group mean was higher (p = 0.0009) than in the T group, but did not differ (p = 0.6802) from the average of the P group. There was no difference between groups P and T (p = 0.2568). Neither in mean blood glucose between the groups (p = 0.4920); serum levels of tacrolimus were similar in PT and T groups (p = 0.7112). There were no histological changes in groups T and C and no hemorrhage in the pancreas of rats in groups P and PT. In group P, there was no edema in 30%, mild edema in 20% and in 50%, moderate; as for inflammatory infiltration, it was moderate in 80% and absent in 20%, and atrophy of the parenchyma was moderate in 60% and severe in 40%. In the PT group, there was edema, inflammatory infiltration or atrophy in the pancreas in all rats. CONCLUSION Treatment with Tacrolimus induced an increase in serum amylase in normal mice, but did not affect blood glucose or the histological pattern of the pancreatic parenchyma. In the presence of pancreatitis induced by L-Arginine tacrolimus induced edema, inflammatory infiltration and more severe atrophy in the pancreatic parenchyma.

Ação da l-arginina na evolução de retalhos cutâneos de ratos sob exposição à nicotina

OBJECTIVE To evaluate whether treatment with L-arginine influences the healing of skin flaps in rats exposed to nicotine. METHODS 40 male Wistar rats weighing 142.4 ± 10.1 g were separated into four groups: GC: treatment with 7.4 pH phosphate buffer, submitted to skin flap and observation for ten days; GN: exposure to nicotine for four weeks, submitted to skin flap and observation for ten days; GA: treatment with 7.4 pH phosphate buffer for four weeks, submitted to skin flap and arginine treatment for ten days; GAN: exposure to nicotine for four weeks, submitted to skin flap and treatment with arginine for ten days. We evaluated: areas of necrosis, re-epithelialization, inflammatory reaction and formation of granulation tissue by HE stain; the total area of deposition and differentiation of collagens I and III by histometry with picrosirius staining; and the scar vascular density by immunohistochemical staining with monoclonal anti-CD34 antibodies. RESULTS The percentages of necrotic areas in GN and GNA were higher (p <0.001) than in GC and GA. In histological scores, collagen deposition, and the percentage of type I collagen, GA and GC were similar to each other (p> 0.05), but higher (p <0.001) than GA and GNA; as for vascular densities, they were lower in GN and GAN (p <0.001) than in GC and GA. CONCLUSION Exposure to nicotine inhibited the effects of arginine and in unexposed mice there was induction of angiogenesis and improvement in the total collagen deposition in the skin flaps.

The influence of nicotine on the population of fibroblasts in cutaneous scars in rats

PURPOSE To study the collagen density and the population of fibroblasts in cutaneous injuries in rats under the influence of nicotine. METHODS The scars of abdominal wounds in rats were analyzed. 2 mg/kg/d of nicotine was administered to the animals in the experiment group and the solution used as a vehicle for the animals in the control group. Treatment was begun seven days prior to surgery and maintained for seven or fourteen days following surgery. The removed scars were prepared for histopathological study. Histological cuts were stained by Sirius Supra red F3BA for collagen analysis and submitted to the examination using the immunohistochemical technique, which enabled us to recognize the population of fibroblasts. RESULTS No significant difference was found in type I collagen density after seven days (p=0.912), nor after fourteen days (p=0.211). The control group had more type III collagen after seven days (p=0.004), but after fourteen days there was no significant difference (p=0,720). The total quantification of collagen, although higher in the control group, was not significantly so at any time during the study (p=0.103 after seven days and p=0.549 after fourteen days). The average of fibroblasts per field was lower after seven days (p=0.0001) and after fourteen days (p=0.0000). CONCLUSION Under the conditions of this experiment, nicotine reduced the fibroblast population without modifying collagen density significantly.

Efeitos da esplenectomia na peritonite causada por lesão traumática do cólon: estudo em ratos

BACKGROUND: Study the effects of splenectomy on the intra-abdominal infection by bowel flora, consequent to a colonic injury in Wistar rats. METHODS: We used 64 animals, 20 for Group A1 (normal with colon lesion left open), 22 for Group A2 (normal with colon lesion sutured) and 22 for Group B (spleen removed). The animals were submitted to a laparatomy through a midline abdominal incision and peritonitis was induced by a colonic lesion in the colon previously distended with 2 ml of saline introduced in the rectum. Bacteriological studies of the abdominal wash obtained with a sterilized swab and microscopic studies of samples of the segment of the sutured colon obtained at 48hrs, 96hrs and on the 12 post op day, were made in each group. All rats were submitted to an autopsy on the day of death or on the 12th post op day when the survivors were sacrificed. RESULTS: Similar bacteria were found in the three groups. E. coli (100%); Enterococcus fecalis (97%); Klebsiela pneumoniae (70%); Citrobacter freundi (70%) and Enterobacter aglomerans (63%). In the first 96 hours the rats without spleen had a lesser inflammatory reaction when compared to the group with spleen. The leading cause of death was generalized peritonitis in the first 96 hours. There was a significant statistical difference in the mortality rate between Group B (80%), Group A2 (no mortality) and Group A1 (35%). CONCLUSION: There was a significant statistical difference in the mortality rate caused by peritonitis between the groups with splenectomy when compared to the group with no splenectomy.

Transplante simultâneo de pâncreas-rim em portador de diabetes mellitus tipo 1 com insuficiência renal crônica: experiência inicial do Hospital Angelina Caron

Pancreatic transplant is the best method for replacing the endocrine function of the gland in patients with type 1 diabetes (DM1). At the end of 2001, more than 17,000 pancreas transplants had been reported to the International Pancreas Transplant Registry. At present, simultaneous pancreaticorenal transplantation is the best treatment for DM1 patients with chronic renal failure. We present the results of simultaneous pancreas-kidney (SPK) transplantation performed in a Brazilian center. METHODS: From 01/2001 to 06/2002, 12 patients with DM1 and associated renal failure received a SPK. Baseline immunosuppression consisted of tacrolimus, mycophenolate mofetil, steroids and induction therapy with basiliximab. RESULTS: With a mean follow-up of 5.7 months (range: 1-18), pancreas and kidney successful rates were respectively 75% and 83%. Survival rate was 83%. There were some major complications including 3 pancreas (25%) and 2 kidney (16%) venous thrombosis and 2 ureteral fistula (16%), but none related to patient death. No episode of rejection occurred in any of the transplanted patients. All patients with successful grafts are insulin-free since transplantation. CONCLUSIONS: These data show that SPK is a safe and effective treatment in the management of DM1 patients with associated renal failure.

Influência do pneumoperitônio nas funções hepática e renal e na liberação de citocinas pró-inflamatórias em modelo experimental de endotoxemia induzida por lipopolissacarídeo de Escherichia coli

BACKGROUND: Videosurgery can bring complications with it´s manipulation and among them the hyperpression into the abdominal cavity is important factor. AIM: To analyze the pneumoperitoneum effects in an endotoxemia experimental model caused by lipopolyssacharide. METHOD: Thirty-two Wistar rats were utilized, from which blood was collected 24 hours prior to the experiment for the initial evaluations of the renal function, the hepatic function and the endotoxic state (total counts and differential of leukocytes, platelet count and dosages of lipopolyssacharide, TNFα, IL6). The rats were then separated into four groups of eight: control groups inoculated with lipopolyssacharide (10 mg/kg via intraperitoneum) and kept for four and 11 hours (C-LPS4 and C-LPS11). Group C-PP, control of pneumoperitoneum by carbon dioxide at 10 mmHg for one hour and kept under observation for six hours. Experiment group (E) in which the rats were inoculated with 10 mg/kg of lipopolyssacharide via intraperitoneum and after four hours were submitted to pneumoperitoneum by carbon dioxide at 10 mmHg for one hour and kept under observation for six hours. At the end of these periods, blood samples were collected for the same initial evaluations. The T-student method was utilized for statistical comparisons of results, with a significance level of 0.05. RESULTS: Compared to the initial values and the control groups, Group E had higher values in the renal function tests (creatinine=0.85 ± 0.24 and urea=106.38 ± 37.61 mg/dL). In the hepatic function tests, there were higher values in the dosages of direct bilirubin (0.34 ± 0.09mg/dL), ALT (390,38 ± 351.88) and TAP (17.01 ± 2.18). The endotoxic state was confirmed by the dosages of IL6 (36.0 ± 11.23 pg/mL), TNFα (518.36 ± 203.39 pg/mL) and by the hematological alterations: reduction of platelets, increased leukocytes and band neutrophils and the detection of circulating endotoxin (0.21 ± 0.08 UE/mL). CONCLUSION: Pneumoperitoneum by carbon dioxide induces increase of TNFα, IL6, worsens the hepatic and renal function in the experimental endotoxemia model induced by lipopolyssacharide.

Ação do pneumoperitônio com dióxido de carbono na translocação bacteriana em ratos

Objective: This study has the purpose of determining if pneumoperitoneum with CO2 develops bacterial translocation in normal rats and in rats submitted to induced colitis. Methods: 60 rats, divided into six groups of 10 animals, were utilized. In the sham group, laparotomy, abdominal organ manipulation, and suture of the wall were performed; the two pneumoperitoneum groups were submitted to pneumoperitoneum for one and three hours, respectively. In the colitis group, colitis was induced, laparotomy, abdominal organ manipulation and suture of the wall were performed. In the two colitis pneumoperitoneum groups colitis was induced plus pneumoperitoneum for one and three hours, respectively. After the animals were sacrificed, kidneys, spleens, livers, and mesenteric lymph nodes were collected. In the animals submitted to induced colitis, 2 cm of colon were resected for histologic analysis. The resected organs were analyzed for the presence of E. coli and Enterococcus faecallis in order to check bacterial translocation. Results: No statistic difference was found in the groups submitted to colitis. The bacterial translocation is no dependent on the genus of bacteria. There is no increase in bacterial translocation due to the duration of pneumoperitoneum (one and three hours) either in normal rats or in those submitted to induced colitis. Conclusion: Pneumoperitoneum induces bacterial translocation in normal rats and increases it in rats submitted to induced colitis.

Resultados do transplante pancreático em um centro brasileiro

BACKGROUND: Pancreatic transplantation (PT) is the only treatment available to keep long term normoglicemic state and insulin independence in type 1 diabetic patients. We present the results of PT performed in a center at the State of Parana. METHODS: From January 2001 until april 2003, 24 patients received simultaneous pancreas-kidney transplant (SPK) and one isolated pancreatic transplant at Hospital Angelina Caron. RESULTS: With a mean follow-up of 8,2 months (range 1-27), the pancreas and kidney success rate was 74%. Patient survival was 76%. Leading cause of pancreas and kidney losses was thrombosis in three pancreas (12%) and two kidneys (8%). No episode of rejection occurred in any of the transplanted patients. All patients with successful grafts are insulin-free since transplantation. CONCLUSIONS : This series confirms that SPK is a highly successful procedure for diabetic patients with renal failure.