Efs Montero

Nitric Oxide Levels in the Intestines of Mice Submitted to Ischemia and Reperfusion: l-Arginine Effects

OBJECTIVE Usually an experimental necrotizing enterocolitis experimental model, we Investigated nitric oxide levels in intestinal tissues of newborn mice with or without l-arginine therapy during sessions of ischemia and reoxygenation. METHODS Twenty-six newborn mice from the Wistar EPM-1 lineage, weighing from 4.5 to 6.2 g, were randomly assigned to three groups: G-I/R, hypoxia and reoxygenation; G-Arg, l-arginine treatment I/R; and G-CTL, controls. G-I/R and G-Arg mice underwent twice a day during their first 3 days of life exposure to gas chambers with 100% CO(2) for 5 minutes at 22 degrees C before reoxygenation with 100% O(2) for another 5 minutes. After 12 hours, all animals were sedated, laparotomized, and had samples of ileum and colon taken and- either formalin fixed histopathologic examinations or frozen to -80 degrees C for estimation of tissue nitric oxide levels. Intestinal injuries were classified according to the criteria of Chiu et al. RESULTS The G-I/R and G-Arg groups showed injuries characteristic of necrotizing enterocolitis (NEC) with an improved structural preservation rate in G-Arg. The concentration of nitric oxide in the Ileum was much higher with G-Arg (16.5 +/- 4.9; P = 0.0019) G-I/R (7.3 +/- 2.0). This effect was not observed in the colon: G-I/R = 10.7 +/- 4.6 versus G-Arg = 15.5 +/- 8.7 (P = .2480). CONCLUSION Supply of L-arginine increased tissue levels of nitricoxide and reduced morphologic intestinal injury among mice undergoing I/R.

Remote ischemic preconditioning on neovascularization and follicle viability on ovary autotransplantation in rats.

PURPOSE Verify the optimum remote vascular occlusion time to reduce ovarian injury in autologous transplants in rats. METHODS Twenty-four adult female rats were assigned to four groups: GC (control group): bilateral oophorectomy followed by ovary transplant; GIPC (ischemic preconditioning group): remote ischemic preconditioning at the iliac artery for 5, 10, and 15 minutes (GIPC-5, GIPC-20, and GIPC-15) previous to bilateral oophorectomy and ovarian transplantation. The right ovary was fixed in the retroperitoneum. Euthanasia was performed 4 days after the surgical procedure. The follicles were counted and classified as developing versus atretic. The immunohistochemical assay identified vascular factor of endothelial growth (VEGF) in the ovarian stroma and assessed the proliferation capacity by means of the Ki-67 in the ovarian follicles. RESULTS Every group showed an inflammatory infiltrate, luteous body, and ovarian follicles in several phases of development. The ischemic preconditioning groups displayed greater amounts of viable ovarian follicles and increased vascularization and vasodilatation than the control group. GIPC-15 showed the highest amount of viable follicles compared to the others (P < .05 GIPC-15 vs GC; GIPC-15 vs GIPC-5). More VEGF-labeled cells were observed in GIPC-10 than the control group (P < .05, GIPC-10 vs GC). The proliferation index assessed by Ki-67 marking showed GC: 80%; GIPC-5: 76%; GIPC-10: 67%; and GIPC-15: 64% (P > .05). CONCLUSIONS The PCI-15 cohort seem to be the most adequate timing to achieve functional support and preservation of a greater number of viable ovarian follicles.

Effect of Remote Ischemic Preconditioning on Rat Estradiol Serum Levels and Follicular Development After Ovarian Transplantation

PURPOSE The purpose of this study was to evaluate estradiol serum levels and follicular development in rats subjected to ovarian autologous transplantation with or without remote ischemic preconditioning (R-IPC). METHODS Seventy-two adult female Wistar EPM-1 rats were distributed into 3 groups: (1) controls; (2) ovarian transplantation; and (3) ovarian transplantation + R-IPC. The groups were divided into subgroups, according to the prefixed date for euthanasia: 24 hours, 48 hours, 72 hours, and 7th postoperative day (PO). R-IPC was performed by clamping the common iliac artery for a 15-minute period of ischemia followed by 15 minutes of reperfusion, before undergoing ovarian transplantation. The graft was fixed to the retroperitoneum with a simple 8-0 prolene thread. Blood samples were collected from the vena cava. For evaluation of follicular development, the ovarian follicles were classified as immature and mature follicles besides corpora lutea. Only the viable follicles and functioning corpora lutea were counted. RESULTS At 72 hours, the R-IPC group showed higher estradiol values than the other groups, which were similar. After 24 hours the mean values were similar among all groups, and at 48 hours the R-IPC group was similar to the transplanted group without IPC. Animals undergoing R-IPC showed superior morphologic aspects, but 7 days after transplantation the morphology was worse in all groups. R-IPC enhanced the number of immature follicles at 48 hours (P > .05) and number of mature follicles from 24 hours to 48 hours after transplantation (P < .01). Functioning corpora lutea number was increased as well. CONCLUSION R-IPC increased the estradiol levels in autologous ovarian transplants associated with better graft morphology and more mature follicles.

Different Intervals of Ischemic Preconditioning on Small Bowel Ischemia-Reperfusion Injury in Rats

PURPOSE The purpose of this study was to establish morphologically the best time of vascular occlusion to induce ischemic preconditioning (IPC) for rat small bowel undergoing ischemia and reperfusion injury. METHODS After approval by the Ethics Committee, 36 EPM-1 young adult Wistar rats from 300-350 g were distributed into 6 groups: sham (S); ischemia and reperfusion (IR), with 50 minutes of cranial mesenteric artery occlusion and 30 minutes of reperfusion; IPC with 1 cycle of 2 minutes (IPC-2), 5 minutes (IPC-5), 10 minutes (IPC-10), or 15 minutes (IPC-15), followed by sustained IR. The animals anesthetized with ketamine (60 mg/kg) and xylazine (10 mg/kg) intramuscular (IM), were maintained on mattress heat, hydrated with saline (80 mL/kg), and injected with 100 IU heparin. Samples of jejunum were stained with hematoxylin and eosin (HE) and classified according to Park et al. Statistical analysis of results was performed using Kruskal-Wallis tests (P < .05). RESULTS The histological evaluation showed no difference between IR and IPC15 rats (5.2 and 5, respectively; P = .84). Greater jejunal injury was observed with IPC15 (5) compared with other groups (IPC2 = 3, P = .03; IPC5 = 3.2, P = .05; IPC10 = 2.8, P = .02, respectively). There was no difference between groups IPC2 x IPC5 x IPC10. CONCLUSION Morphologically, IPC with short times promoted greater intestinal protection against the IR lesion in rats.

Inhibition of mouse and rat lymphoproliferation by gangliosides

Our previous study have demonstrated that Glycosphingolipids (GSLs) have an immunosuppressive effect on murine lymphoproliferation and IL-2 production. In the present study we examined the effect of a pool of Gangliosides (Gang) on spleen lymphocyte proliferation from either isogeneic strains of Wistar rats or BALB/c mice. Two hundred-fifty grams adult female isogeneic Wistar rats and 8-week-old BALB/c mice were used. The animals were sacrificed and the spleen harvested aseptically for cellular assays. Spleen cells suspensions were obtained by homogenization in RPMI 1640 with a loose tissue grinder. After washing, the cells were suspended in RPMI 1640 supplemented. Cell viability was measured by Trypan blue exclusion. Cells were cultured in triplicate using increasing concentrations of Gang (1; 2; 5; 10; 15; 20 mg/well) and in the presence of Concanavalin A. The cells were incubated for 48 hours and were pulsed with [3H] thymidine 18 hours prior to harvesting on glass fiber paper for counting in a b-counter. Data were presented as rate of inhibition, as previously described. At concentrations 1 and 2 mg/well, Gang stimulated lymphoproliferation (30% and 50%, rats and mice respectively), while at concentration from 5 to 20 mg/well an increasing inhibition was observed for spleen cells from both mouse and rat (from 40% up to 80%). In preliminary studies we observed inhibition of mixed lymphocyte reaction on spleen cells from rats treated with Gang for 10 days (data not shown). Our data suggest that Gang may be investigated as a immunosuppressive drug in organ transplantation.

Efeitos do pneumoperitônio no rato

Effects of pneumoperitoneum in rats. The effects of different pneumoperitoneal pressures on kidney and liver functions and on arterial gasometry in rats are provided. A sample of twenty male rats weighing from 350 to 400 grams and distributed into two groups of ten animals was used. Each animal served as experimental control from which blood samples were collected before introducing CO2 into the peritoneal cavity. Samples underwent pressures of 4 and 6 mm Hg during one hour. Results show that increase in intra-abdominal pressure by CO2 in rats causes changes in their arterial gasometry, liver and kidney. However, no liver or kidney lesions during the above mentioned time were reported.

Gangliosideos - estudo do colágeno e da resposta inflamatória no processo cicatricial

Objetivo: Investigar o efeito dos gangliosideos sobre a infiltracao sequencial de leucocitos e fibroblastos durante o processo de cicatrizacao usando um modelo de cicatrizacao da pele em ratos. Metodo: Foram utilizados 12 ratas EPM - 1 Wistar, com peso medio de 200 gramas e 4 meses de idade. Os animais procederam do Centro de Desenvolvimento de Pesquisa Experimental em Medicina e Biologia e foram mantidos por 5 dias para adaptacao no bioterio setorial da disciplina de Tecnica Operatoria e Cirurgia Experimental da UNIFESP-EPM, recebendo agua e racao propria para a especie. O protocolo anestesico utilizado foi uma associacao de Cetamina (60mg.kg-1) e Xilazina (10mg.kg-1), por via intramuscular. Em seguida, realizava-se uma incisao longitudinal com 7 cm de extensao, na regiao dorsal paravertebral, interessando pele e tela subcutânea que foi fechada com pontos separados, com fio de prolene 7-0 e agulha triangular. As ratas foram distribuidas em dois grupos a saber: grupo experimento, que recebeu 3mg.kg-1.dia-1 de gangliosideos, e um grupo controle, que recebeu veiculo, ambos por via intramuscular durante 14 dias consecutivos. No 7o e 14o dias de pos-operatorio foram ressecados fragmentos da pele e tela subcutânea para analise histologica, com a coloracao de Tricromio de Masson e Hematoxilina - Eosina. Resultados: As amostras apresentaram a mesma quantidade de colageno em ambos os grupos mostrando que nao houve inibicao dos fibroblastos. Entretanto, a infiltracao leucocitaria foi retardada no grupo experimento quando comparado ao grupo controle. Conclusao: A alteracao encontrada no processo cicatricial foi devida a um retardo na resposta inflamatoria e nao a uma inibicao fibroblastos.